Inhibitors of Human EZH2 and Methods of Use Thereof

ABSTRACT

The invention relates to determining the presence of an EZH2 gene mutation in a sample from a subject and inhibition of wild-type and certain mutant forms of human histone methyltransferase EZH2, the catalytic subunit of the PRC2 complex which catalyzes the mono-through tri-methylation of lysine 27 on histone H3 (H3-K27). In one embodiment the inhibition is selective for the mutant form of the EZH2, such that trimethylation of H3-K27, which is associated with certain cancers, is inhibited. The methods can be used to treat cancers including follicular lymphoma and diffuse large B-cell lymphoma (DLBCL). Also provided are methods for identifying small molecule selective inhibitors of the mutant forms of EZH2 and also methods for determining responsiveness to an EZH2 inhibitor in a subject.

RELATED REFERENCE

This application is a continuation of U.S. patent application Ser. No.13/230,703, filed Sep. 12, 2011, which claims priority to, and thebenefit of, U.S. Ser. No. 61/381,684, filed Sep. 10, 2010, the contentsof each of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

This invention relates to inhibition of wild-type and certain mutantforms of human histone methyltransferase EZH2, the catalytic subunit ofthe PRC2 complex which catalyzes the mono- through tri-methylation oflysine 27 on histone H3 (H3-K27), methods for treating cancers includingfollicular lymphoma and diffuse large B-cell lymphoma (DLBCL) andmethods for determining responsiveness to an EZH2 inhibitor in asubject.

BACKGROUND

In eukaryotic cells DNA is packaged with histones to form chromatin.Approximately 150 base pairs of DNA are wrapped twice around an octamerof histones (two each of histones 2A, 2B, 3 and 4) to form a nucleosome,the basic unit of chromatin. Changes in the ordered structure ofchromatin can lead to alterations in transcription of associated genes.This process is highly controlled because changes in gene expressionpatterns can profoundly affect fundamental cellular processes, such asdifferentiation, proliferation and apoptosis. Control of changes inchromatin structure (and hence of transcription) is mediated by covalentmodifications to histones, most notably of their N-terminal tails. Thesemodifications are often referred to as epigenetic because they can leadto heritable changes in gene expression, but they do not affect thesequence of the DNA itself. Covalent modifications (for example,methylation, acetylation, phosphorylation and ubiquitination) of theside chains of amino acids are enzymatically mediated.

The selective addition of methyl groups to specific amino acid sites onhistones is controlled by the action of a unique family of enzymes knownas histone methyltransferases (HMTs). The level of expression of aparticular gene is influenced by the presence or absence of one or moremethyl groups at a relevant histone site. The specific effect of amethyl group at a particular histone site persists until the methylgroup is removed by a histone demethylase, or until the modified histoneis replaced through nucleosome turnover. In a like manner, other enzymeclasses can decorate DNA and histones with other chemical species, andstill other enzymes can remove these species to provide control of geneexpression.

The orchestrated collection of biochemical systems behindtranscriptional regulation must be tightly controlled in order for cellgrowth and differentiation to proceed optimally. Disease states resultwhen these controls are disrupted by aberrant expression and/or activityof the enzymes responsible for DNA and histone modification. In humancancers, for example, there is a growing body of evidence to suggestthat dysregulated epigenetic enzyme activity contributes to theuncontrolled cell proliferation associated with cancer as well as othercancer-relevant phenotypes such as enhanced cell migration and invasion.Beyond cancer, there is growing evidence for a role of epigeneticenzymes in a number of other human diseases, including metabolicdiseases (such as diabetes), inflammatory diseases (such as Crohn'sdisease), neurodegenerative diseases (such as Alzheimer's disease), andcardiovascular diseases. Therefore, selectively modulating the aberrantaction of epigenetic enzymes holds great promise for the treatment of arange of diseases.

Histone Methyltransferase EZH2

Polycomb group (PcG) and trithorax group (trxG) proteins are known to bepart of the cellular memory system. Francis et al. (2001) Nat Rev MolCell Biol 2:409-21; Simon et al. (2002) Curr Opin Genet Dev 12:210-8.Both groups of proteins are involved in maintaining the spatial patternsof homeotic box (Hox) gene expression, which are established early inembryonic development by transiently expressed segmentation genes. Ingeneral, PcG proteins are transcriptional repressors that maintain the“off state,” and trxG proteins are transcriptional activators thatmaintain the “on state.” Because members of PcG and trxG proteinscontain intrinsic histone methyltransferase (HMTase) activity, PcG andtrxG proteins may participate in cellular memory through methylation ofcore histones. Beisel et al. (2002) Nature 419:857-62; Cao et al. (2002)Science 298:1039-43; Czermin et al. (2002) Cell 111:185-96; Kuzmichev etal. (2002) Genes Dev 16:2893-905; Milne et al. (2002) Mol Cell10:1107-17; Muller et al. (2002) Cell 111:197-208; Nakamura et al.(2002) Mol Cell 10:1119-28.

Biochemical and genetic studies have provided evidence that DrosophilaPcG proteins function in at least two distinct protein complexes, thePolycomb repressive complex 1 (PRC 1) and the ESC-E(Z) complex (alsoknown as Polycomb repressive complex 2 (PRC2)), although thecompositions of the complexes may be dynamic. Otte et al. (2003) CurrOpin Genet Dev 13:448-54. Studies in Drosophila (Czermin et al. (supra);Muller et al. (supra)) and mammalian cells (Cao et al. (supra);Kuzmichev et al. (supra)) have demonstrated that the ESC-E(Z)/EED-EZH2(i.e., PRC2) complexes have intrinsic histone methyltransferaseactivity. Although the compositions of the complexes isolated bydifferent groups are slightly different, they generally contain EED,EZH2, SUZ12, and RbAp48 or Drosophila homologs thereof. However, areconstituted complex comprising only EED, EZH2, and SUZ12 retainshistone methyltransferase activity for lysine 27 of histone H3. U.S.Pat. No. 7,563,589 (incorporated by reference).

Of the various proteins making up PRC2 complexes, EZH2 (Enhancer ofZeste Homolog 2) is the catalytic subunit. The catalytic site of EZH2 inturn is present within a SET domain, a highly conserved sequence motif(named after Su(var)3-9, Enhancer of Zeste, Trithorax) that is found inseveral chromatin-associated proteins, including members of both theTrithorax group and Polycomb group. SET domain is characteristic of allknown histone lysine methyltransferases except the H3-K79methyltransferase DOTI.

In addition to Hox gene silencing, PRC2-mediated histone H3-K27methylation has been shown to participate in X-inactivation. Plath etal. (2003) Science 300:131-5; Silva et al. (2003) Dev Cell 4:481-95.Recruitment of the PRC2 complex to Xi and subsequent trimethylation onhistone H3-K27 occurs during the initiation stage of X-inactivation andis dependent on Xist RNA. Furthermore, EZH2 and its associated histoneH3-K27 methyltransferase activity was found to mark differentially thepluripotent epiblast cells and the differentiated trophectoderm. Erhardtet al. (2003) Development 130:4235-48).

Consistent with a role of EZH2 in maintaining the epigeneticmodification patterns of pluripotent epiblast cells, Cre-mediateddeletion of EZH2 results in loss of histone H3-K27 methylation in thecells. Erhardt et al. (supra). Further, studies in prostate and breastcancer cell lines and tissues have revealed a strong correlation betweenthe levels of EZH2 and SUZ12 and the invasiveness of these cancers(Bracken et al. (2003) EMBO J 22:5323-35; Kirmizis et al. (2003) MolCancer Ther 2:113-21; Kleer et al. (2003) Proc Natl Acad Sci USA100:11606-11; Varambally et al. (2002) Nature 419:624-9), indicatingthat dysfunction of the PRC2 complex may contribute to cancer.

Recently, somatic mutations of tyrosine 641 (Y641F, Y641N, Y641S andY641H) of EZH2 were reported to be associated with follicular lymphoma(FL) and the germinal center B cell-like (GCB) subtype of diffuse largeB-cell lymphoma (DLBCL). Morin et al. (2010) Nat Genet. 42:181-5. In allcases, occurrence of the mutant EZH2 gene was found to be heterozygous,and expression of both wild-type and mutant alleles was detected in themutant samples profiled by transcriptome sequencing. It was alsodemonstrated that all of the mutant forms of EZH2 could be incorporatedinto the multi-protein PRC2 complex, but that the resulting complexeslacked the ability to catalyze methylation of the H3-K27 equivalentresidue of a peptidic substrate. Hence, it was concluded that thedisease-associated changes at Tyr641 of EZH2 resulted in loss offunction with respect to EZH2-catalyzed H3-K27 methylation.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to modulating the activityof the wild-type and mutant histone methyltransferase EZH2, thecatalytic subunit of the PRC2 complex which catalyzes the mono- throughtri-methylation of lysine 27 on histone H3 (H3-K27). For example, thepresent invention relates to inhibiting the activity of certain mutantforms of EZH2. The mutant forms of EZH2 include a substitution ofanother amino acid residue for tyrosine 641 (Y641, also Tyr641) ofwild-type EZH2.

Another aspect of the present invention relates to determining apatient's responsiveness to an EZH2 inhibitor according to thedimethylated H3-K27me2 level, or preferably according to the levels ofdimethylated H3-K27me2 and trimethylated H3-K27me3. For example, cellswith low or undetectable level of dimethylated H3-K27me2 or cells withlow ratio of H3-K27me2/me3 are much more responsive to theanti-proliferative effect of an EZH2 inhibitor than cells with the moretypical higher H3-K27me2/me3 ratio.

An aspect of the invention is a method of inhibiting in a subjectconversion of H3-K27 to trimethylated H3-K27. The method comprises thestep of administering to a subject expressing a Y641 mutant of EZH2 atherapeutically effective amount of an inhibitor of EZH2, wherein theinhibitor inhibits histone methyltransferase activity of EZH2, therebyinhibiting conversion of H3-K27 to trimethylated H3-K27 in the subject.

In this and other aspects of the invention, in one embodiment theinhibitor inhibits histone methyltransferase activity of the Y641 mutantof EZH2.

In this and other aspects of the invention, in one embodiment theinhibitor selectively inhibits histone methyltransferase activity of theY641 mutant of EZH2.

In this and other aspects of the invention, in one embodiment the Y641mutant of EZH2 is selected from the group consisting of Y641F, Y641H,Y641N, and Y641S.

In this and other aspects of the invention, in one embodiment theinhibitor of EZH2 is S-adenosyl-L-homocysteine or a pharmaceuticallyacceptable salt thereof.

In this and other aspects of the invention, in one embodiment theinhibitor of EZH2 is Compound 75

or a pharmaceutically acceptable salt thereof.

An aspect of the invention is a method of inhibiting in a subjectconversion of H3-K27 to trimethylated H3-K27. The method comprises thesteps of performing an assay to detect a Y641 mutant of EZH2 in a samplefrom a subject; and administering to a subject expressing a Y641 mutantof EZH2 a therapeutically effective amount of an inhibitor of EZH2,wherein the inhibitor inhibits histone methyltransferase activity ofEZH2, thereby inhibiting conversion of H3-K27 to trimethylated H3-K27 inthe subject.

In this and other aspects of the invention, in one embodiment,performing the assay to detect the Y641 mutant of EZH2 includeswhole-genome resequencing or target region resequencing that detects anucleic acid encoding the Y641 mutant of EZH2.

In this and other aspects of the invention, in one embodiment,performing the assay to detect the Y641 mutant of EZH2 includescontacting the sample with an antibody that binds specifically to apolypeptide or fragment thereof characteristic of the Y641 mutant ofEZH2.

In this and other aspects of the invention, in one embodiment,performing the assay to detect the Y641 mutant of EZH2 includescontacting the sample under highly stringent conditions with a nucleicacid probe that hybridizes to a nucleic acid encoding a polypeptide orfragment thereof characteristic of the Y641 mutant of EZH2.

An aspect of the invention is a method of inhibiting conversion ofH3-K27 to trimethylated H3-K27. The method comprises the step ofcontacting a Y641 mutant of EZH2 with a histone substrate comprisingH3-K27 and an effective amount of an inhibitor of EZH2, wherein theinhibitor inhibits histone methyltransferase activity of EZH2, therebyinhibiting conversion of H3-K27 to trimethylated H3-K27.

An aspect of the invention is a method of identifying a subject as acandidate for treatment with an inhibitor of EZH2. The method comprisesthe steps of performing an assay to detect a Y641 mutant of EZH2 in asample from a subject; and identifying a subject expressing a Y641mutant of EZH2 as a candidate for treatment with an inhibitor of EZH2,wherein the inhibitor inhibits histone methyltransferase activity ofEZH2.

An aspect of the invention is a method identifying an inhibitor of aY641 mutant of EZH2. The method comprises the steps of combining anisolated Y641 mutant of EZH2 with a histone substrate, a methyl groupdonor, and a test compound, wherein the histone substrate comprises aform of H3-K27 selected from the group consisting of unmethylatedH3-K27, monomethylated H3-K27, dimethylated H3-K27, and any combinationof thereof; and performing an assay to detect methylation of H3-K27 inthe histone substrate, thereby identifying the test compound as aninhibitor of the Y641 mutant of EZH2 when methylation of H3-K27 in thepresence of the test compound is less than methylation of H3-K27 in theabsence of the test compound.

In one embodiment, performing the assay to detect methylation of H3-K27in the histone substrate comprises measuring incorporation of labeledmethyl groups.

In one embodiment, the labeled methyl groups are isotopically labeledmethyl groups.

In one embodiment, performing the assay to detect methylation of H3-K27in the histone substrate comprises contacting the histone substrate withan antibody that binds specifically to trimethylated H3-K27.

An aspect of the invention is a method of identifying an inhibitor of aY641 mutant of EZH2. The method comprises the steps of combining anisolated Y641 mutant of EZH2 with a histone substrate, a methyl groupdonor, and a test compound, wherein the histone substrate comprises aform of H3-K27 selected from the group consisting of unmethylatedH3-K27, monomethylated H3-K27, dimethylated H3-K27, and any combinationthereof; and performing an assay to detect formation of trimethylatedH3-K27 in the histone substrate, thereby identifying the test compoundas an inhibitor of the Y641 mutant of EZH2 when formation oftrimethylated H3-K27 in the presence of the test compound is less thanformation of trimethylated H3-K27 in the absence of the test compound.

In one embodiment, performing the assay to detect formation oftrimethylated H3-K27 in the histone substrate comprises measuringincorporation of labeled methyl groups.

In one embodiment, the labeled methyl groups are isotopically labeledmethyl groups.

In one embodiment, performing the assay to detect formation oftrimethylated H3-K27 in the histone substrate comprises contacting thehistone substrate with an antibody that binds specifically totrimethylated H3-K27.

An aspect of the invention is a method of identifying a selectiveinhibitor of a Y641 mutant of EZH2. The method comprises the steps ofcombining an isolated Y641 mutant of EZH2 with a histone substrate, amethyl group donor, and a test compound, wherein the histone substratecomprises a form of H3-K27 selected from the group consisting ofmonomethylated H3-K27, dimethylated H3-K27, and a combination ofmonomethylated H3-K27 and dimethylated H3-K27, thereby forming a testmixture; combining an isolated wild-type EZH2 with a histone substrate,a methyl group donor, and a test compound, wherein the histone substratecomprises a form of H3-K27 selected from the group consisting ofmonomethylated H3-K27, dimethylated H3-K27, and a combination ofmonomethylated H3-K27 and dimethylated H3-K27, thereby forming a controlmixture; performing an assay to detect trimethylation of the histonesubstrate in each of the test mixture and the control mixture;calculating the ratio of (a) trimethylation with the Y641 mutant of EZH2and the test compound (M+) to (b) trimethylation with the Y641 mutant ofEZH2 without the test compound (M−); calculating the ratio of (c)trimethylation with wild-type EZH2 and the test compound (WT+) to (d)trimethylation with wild-type EZH2 without the test compound (WT−);comparing the ratio (a)/(b) with the ratio (c)/(d); and identifying thetest compound as a selective inhibitor of the Y641 mutant of EZH2 whenthe ratio (a)/(b) is less than the ratio (c)/(d).

An aspect of the invention is a method of treating cancer. The methodcomprises the step of administering to a subject having a cancerexpressing a Y641 mutant of EZH2 a therapeutically effective amount ofan inhibitor of EZH2, wherein the inhibitor inhibits histonemethyltransferase activity of EZH2, thereby treating the cancer.

In this and other aspects of the invention, in one embodiment the canceris selected from the group consisting of follicular lymphoma and diffuselarge B-cell lymphoma (DLBCL) of germinal center B cell-like (GCB)subtype.

An aspect of the invention is a method of treating cancer. The methodcomprises the step of administering to a subject having a cancerexpressing a Y641 mutant of EZH2 a therapeutically effective amount ofan inhibitor of EZH2, wherein the inhibitor selectively inhibits histonemethyltransferase activity of the Y641 mutant of EZH2, thereby treatingthe cancer.

An aspect of the invention is a method of treating cancer. The methodcomprises the steps of performing an assay to detect a Y641 mutant ofEZH2 in a sample comprising cancer cells from a subject having a cancer;and administering to a subject expressing a Y641 mutant of EZH2 atherapeutically effective amount of an inhibitor of EZH2, wherein theinhibitor inhibits histone methyltransferase activity of EZH2, therebytreating the cancer.

Another aspect of the invention is a method for determiningresponsiveness to an EZH2 inhibitor in a subject. In one embodiment themethod includes isolating a tissue sample from the subject; detecting adimethylation (me2) level of H3-K27 in the tissue sample; comparing thedimethylation (me2) level to a control dimethylation (me2) level; andidentifying the subject is responsive to said EZH2 inhibitor when thedimethylation (me2) level is absent or lower than the controldimethylation (me2) level. In one embodiment, the method furtherincludes detecting a trimethylation (me3) level of H3-K27 in the tissuesample; comparing the trimethylation (me3) level to a controltrimethylation (me3) level and the dimethylation (me2) level to acontrol dimethylation (me2) level; and identifying said subject isresponsive to the EZH2 inhibitor when the trimethylation (me3) level issame as or higher than the control trimethylation (me3) level and thedimethylation (me2) level is absent or lower than the controldimethylation (me2) level. In another embodiment, the method furtherincludes obtaining a ratio of the dimethylation (me2) level to thetrimethylation (me3) level of H3-K27 in the tissue sample; obtaining acontrol ratio of the control dimethylation (me2) level to the controltrimethylation (me3) level; comparing the ratio to the control ratio;and identifying the subject is responsive to said EZH2 inhibitor whensaid ratio is lower than said control ratio. In a preferred embodiment,the subject has cancer. In one embodiment, the cancer is a follicularlymphoma. Alternatively, the cancer is a diffuse large B-cell lymphoma(DLBCL). In another preferred embodiment, the subject expresses a Y641mutant EZH2. In a preferred embodiment, the Y641 mutant is Y641F, Y641H,Y641N or Y641S.

An aspect of the invention is method for selecting a treatment for asubject having a cancer. The method includes determining responsivenessof the subject to an EZH2 inhibitor by the dimethylated H3-K27 level orpreferably by the levels of dimethylated H3-K27 and trimethlated H3-K27;and providing the EZH2 inhibitor to the subject when the subject isresponsive to the EZH2 inhibitor. In one embodiment, the cancer is afollicular lymphoma. Alternatively, the cancer is a diffuse large B-celllymphoma (DLBCL). In another preferred embodiment, the subject expressesa Y641 mutant EZH2. In a preferred embodiment, the Y641 mutant is Y641F,Y641H, Y641N or Y641S.

An aspect of the invention is Compound 75

or a pharmaceutically acceptable salt thereof.

An aspect of the invention is a pharmaceutical composition comprisingCompound 75

or a pharmaceutically acceptable salt thereof.

An aspect of the invention is the use of Compound 75

or a pharmaceutically acceptable salt thereof in the treatment offollicular lymphoma.

An aspect of the invention is the use of Compound 75

or a pharmaceutically acceptable salt thereof in the treatment ofdiffuse large B-cell lymphoma (DLBCL).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods and examples areillustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is two graphs establishing that B-cell lymphoma-associatedmutants of EZH2 are active histone methyltransferases. In vitromethyltransferase activity of PRC2 complexes containing wild-type andvarious Y641 mutants of EZH2 was measured as (A) methyl transferreactions using a peptide (H3 21-44) as substrate, and (B) methyltransfer reactions using avian nucleosomes as substrate. Symbols:wild-type (), Y641F (∘), Y641H (□), Y641N (▪), and Y641S (▴). CPM iscounts per minute, referring to scintillation counting as a result of ³Hradiation.

FIG. 2 is four graphs establishing that PRC2 complexes containing mutantEZH2 preferentially catalyze di- and tri-methylation of histone H3-K27.(A) Methyltransferase activity of mutant and wild-type (WT) complexes onunmethylated peptide (open bars), monomethylated peptide (hashed bars),and dimethylated peptide (closed bars). (B) Affinity for peptidesubstrates as judged by K_(1/2) is similar across all peptidemethylation states for PRC2 complexes containing wild-type (∘), Y641F(), Y641H (□), Y641N (▪), and Y641S (▴) EZH2. Note that the variationin K_(1/2) values across all substrates and all enzyme forms is lessthan 3.5-fold. For any particular methylation state of substrate thevariation in K_(1/2) value is less than 2-fold. (C) Enzyme turnovernumber (k_(cat)) varies with substrate methylation status in opposingways for WT and Y641 mutants of EZH2. The k_(cat) decreases withincreasing K27 methylation states for wild-type (∘), but increases forY641F (), Y641H (□), □Y641N (▪), and Y641S (▴) mutants of EZH2. (D)Catalytic efficiency (k_(cat)/K_(1/2)) decreases with increasing K27methylation states for wild-type (∘), but increases for Y641F (),Y641H(□), Y641N (▪), and Y641S (▴) mutants of EZH2. In panels B-D, thelines drawn to connect the data points are not intended to imply anymathematical relationship; rather, they are merely intended to serve asvisual aides.

FIG. 3A is a trio of graphs depicting predicted relative levels ofH3-K27me3 (top panel), H3-K27me2 (middle panel), and H3-K27me1 (bottompanel) for cells containing different EZH2 mutants. Simulations wereperformed using a coupled enzyme steady state velocity equation and thesteady state kinetic parameters shown in Table 1. All values arerelative to the homozygous WT EZH2-containing cells and assumesaturating concentrations of intracellular SAM, relative to Km andintracellular nucleosome concentrations similar to Km.

FIG. 3B is a series of Western blot analyses of relative patterns ofH3-K27 methylation status for lymphoma cell lines homozygous for WTEZH2, or heterozygous for the indicated EZH2 Y641 mutation. Panels fromtop to bottom depict the results of probing with antibodies specific forthe following: total EZH2; H3-K27me3; H3-K27me2; H3-K27me1; and totalhistone H3 as loading control.

FIG. 4 depicts selected proposed mechanisms leading to aberrantly highlevels of trimethylation on histone H3-K27 in cancer. These include: a)mutation of Y641 in EZH2 resulting in a change in substrate preferencefrom the nonmethylated to the mono- and di-methylated histone H3-K27; b)overexpression of EZH2; c) mutations in UTX that inactivate enzymefunction, causing a decrease in demethylation of H3-K27me3; and d)overexpression of the PRC2 complex subunit PHF19/PCL3 that leads toincreases in recruitment of the PRC2 complex to specific genes and anincrease in histone H3-K27 trimethylation. In all four models thealteration leads to aberrant histone H3-K27 trimethylation in theproximal promoter regions of genes resulting in transcriptionalrepression of key genes in cancer.

FIG. 5 depicts a SDS-PAGE gel showing that the expression levels of eachof the five-component PRC2 complexes are similar with mutant andwild-type EZH2.

FIG. 6 is a pair of tables showing that mutant and wild-type (WT) PRC2complexes display strong substrate preference for H3-K27-containingpeptides. Each enzyme was tested against a panel of overlapping 15-merpeptides covering all of H3 and H4. Activity was measured as velocity(CPM per minute), and the reported value represents the mean of twoindependent determinations for each reaction. For all the complexes themost favored peptide was H3:16-30. WT complex had greater than 6-foldmore activity against this peptide than any of the mutant complexes.

FIG. 7 is a graph depicting inhibitory potency ofS-adenosyl-L-homocysteine (SAH) against EZH2 WT and Y641 mutants ofEZH2. The X axis shows log concentration of SAH; the Y axis showspercent inhibition.

FIG. 8 is a graph depicting inhibitory potency of Compound 75 againstEZH2 WT and Y641 mutants of EZH2. The X axis shows log concentration ofCompound 75; the Y axis shows percent inhibition.

FIG. 9 depicts a Western Blot analysis of relative levels of H3-K27me1,me2 and me3 in a cell line pane, including multiple DLBCL linesexpressing WT or Y641 mutatnt EZH2. a) Histones were extracted from thecell lines shown, fractionated by SDS-PAGE on a 4-20% gel, transferredto nitrocellulose membranes, and probed with antibodies to Histone H3,H3-K27me1, me2, or me3. EZH2 levels were determined by preparing wholecell lysates from the cell lines shown, treating as above and probingwith an antibody to EZH2; b) Histones were extracted from the cell linesshown and treated as above, except EZH2 levels were not determined.

FIG. 10 depicts an immunocytochemistry analysis of H3 and H3-K27me3levels in a panel of WT and Y641 mutant lymphoma cell lines. Cellpellets from the indicated cell lines were fixed and embedded inparaffin. Slides were prepared and levels of H3 and H3-K27me3 wereevaluated by immunocytochemistry using antibodies to histone H3, orH3-K27me3.

FIG. 11 depicts an immunocytochemistry analysis of H3 and H3-K27me2levels in a panel of WT and Y641 mutant lymphoma cell lines. Cellpellets from the indicated cell lines were fixed and embedded inparaffin. Slides were prepared and levels of H3 and H3-K27me2 wereevaluated by immunocytochemistry using antibodies to histone H3, orH3-K27me2.

FIG. 12 is a graph depicting the inhibition of global H3-K27me3 levelsby EZH2 inhibitor treatment in Y641 mutant WSU-DLCL2 cells. WSU-DLCL2cells were treated for 4 days with the indicated concentrations of EZH2inhibitor A or B. Following compound treatment, histones were extracted,fractionated by SDS-PAGE on a 4-20% gel, transferred to nitrocellulosemembranes, and probed with antibodies to Histone H3, or H3-K27me3.

FIG. 13 is a graph showing that the EZH2 inhibitors can blockproliferation of a Y641 mutant WSU-DLCL2 cells, but has little effect onnon Y641 mutant OCI-LY19 cells. Cells were incubated in the presence ofincreasing concentrations of EZH2 inhibitor A or B for eleven days.Vehicle treated (DMSO) cells were included as controls. Cell number andviability was determined using the Guava Viacount assay in a GuavaEasyCyte Plus instrument. Cells were split and media and compound wasreplenished every 3-4 days.

FIG. 14 is a graph showing the presence of an EZH2 (Y641) mutationand/or high H3-K27me3 and low H3-K27me2 levels predict sensitivity toEZH2 inhibitors. Cell lines were maintained in the presence ofincreasing concentrations of one EZH2 inhibitor up to 25 μM. Viablecells counts were used to derive IC90 values after 11 days of treatment.Results are plotted with cell lines segregated according to EZH2mutational status (A), or segregated according to H3-K27me2 andH3-K27me3 levels (B). In both plots, the line shows the average IC90values from the indicated cell line group.

DETAILED DESCRIPTION

Chromatin structure is important in gene regulation and epigeneticinheritance. Post-translational modifications of histones are involvedin the establishment and maintenance of higher-order chromatinstructure; for example, the tails of certain core histones are modifiedby acetylation, methylation, phosphorylation, ribosylation and/orubiquitination.

EZH2 is a histone methyltransferase that is the catalytic subunit of thePRC2 complex which catalyzes the mono- through tri-methylation of lysine27 on histone H3 (H3-K27). Histone H3-K27 trimethylation is a mechanismfor suppressing transcription of specific genes that are proximal to thesite of histone modification. This trimethylation is known to be acancer marker with altered expression in cancer, such as prostate cancer(see, e.g., U.S. Patent Application Publication No. 2003/0175736;incorporated herein by reference in its entirety). EZH2 belongs to thePolycomb group protein family (PcG). The polycomb group proteins help inmaintaining cellular identity by transcriptional repression of targetgenes. Jacobs et al. (1999) Semin Cell Dev Biol 10(2):227-35; Jacobs etal. (2002) Biochim Biophys Acta 1602(2):151-61. DNA microarraysidentified EZH2 as being up-regulated in hormone-refractory metastaticprostate cancer. Dhanasekaran et al. (2001) Nature 412(6849):822-6;Varambally et al. (2002) Nature 419(6907):624-9. EZH2 is up-regulated inaggressive breast tumors and is a mediator of a pro-invasive phenotype.Kleer et al. (2003) Proc Natl Acad Sci USA 100(20):11606-11.Overexpression of EZH2 in immortalized human mammary epithelial celllines promotes anchorage-independent growth and cell invasion. Kleer etal. (supra). EZH2-mediated cell invasion required an intact SET domainand histone deacetylase activity. Previous studies provided evidence fora functional link between dysregulated EZH2 expression, transcriptionalrepression, and neoplastic transformation. Varambally et al. (supra);Kleer et al (supra).

An aspect of the present invention relates to inhibiting the activity ofEZH2, including certain mutant forms of EZH2. In one embodiment thepresent invention relates to inhibiting selectively the activity ofcertain mutant forms of EZH2.

Point mutations of the EZH2 gene at a single amino acid residue (Tyr641,herein referred to as Y641) of EZH2 have been reported to be linked tosubsets of human B-cell lymphoma. Morin et al. (2010) Nat Genet.42(2):181-5. In particular, Morin et al. reported that somatic mutationsof tyrosine 641 (Y641F, Y641H, Y641N, and Y641S) of EZH2 were associatedwith follicular lymphoma (FL) and the germinal center B cell-like (GCB)subtype of diffuse large B-cell lymphoma (DLBCL). The mutant allele isalways found associated with a wild-type allele (heterozygous) indisease cells, and the mutations were reported to ablate the enzymaticactivity of the PRC2 complex for methylating an unmodified peptidesubstrate.

It has now been unexpectedly discovered that the wild-type (WT) EZH2enzyme displays greatest catalytic efficiency (kcat/K) for the zero- tomono-methylation reaction of H3-K27 and lesser efficiency for subsequent(mono- to di- and di- to tri-methylation) reactions; whereas, in starkcontrast, the disease-associated Y641 mutations display very limitedability to perform the first methylation reaction but have enhancedcatalytic efficiency for the subsequent reactions relative to wild-typeenzyme. These results imply that the malignant phenotype of diseaseexploits the combined activities of a H3-K27 mono-methylating enzyme(PRC2 containing WT EZH2 or EZH1) together with PRC2 containing mutantEZH2 for augmented conversion of H3-K27 to the tri-methylated form(H3-K27me3).

While not intending to be bound by any one theory, it is hypothesizedthat the mutation of Y641 to phenylalanine (F), histidine (H),asparagine (N), or serine (S) in EZH2 may facilitate multiple rounds ofH3-K27 methylation by impacting the H-bonding pattern and/or stericcrowding in the active site of the enzyme-bisubstrate ternary complex,affecting the formation of a proper water channel for deprotonation ofthe reacting lysine. Zhang et al. (2008) Proc Natl Acad Sci USA105:5728-32. This inference is drawn by analogy to the crystallographicand molecular dynamic simulation results seen for tyrosine mutation inthe related protein lysine methyltransferases LSMT, Dim-5 and SET7/9.

For example, when tyrosine 245 of recombinant SET7/9 was mutated toalanine a change in substrate specificity was observed. Dillon et al.(2005) Genome Biol 6:227. The ability of the Y245A mutant SET7/9 tomethylate an unmodified 20-residue peptide, representing the sequencesurrounding H3-K4, was reduced to ca. 20% of WT enzyme. Xiao et al.(2003) Nature 421:652-6. At the same time, the ability of the Y245Amutant to further methylate H3-K4me1 and H3-K4me2 peptides was greatlyaugmented (ca. 7-fold and 5-fold, respectively) relative to WT enzyme.In contrast to the instant disclosure in respect of mutations of Y641 ofEZH2, however, mutation of SET7/9 Y245 to phenylalanine did not enhancemono-to-di nor di-to-tri methylation of the peptide; rather, the Y245Fmutant of SET7/9 displayed minimal catalytic activity for all peptidicsubstrates. Similarly, the wild-type enzyme G9a can dimethylate H3-K9but is unable to perform the di-to-trimethylation reaction. Yet, whentyrosine 1067 of G9a (analogous to Y641 of EZH2) is mutated tophenylalanine, the enzyme now gains the ability to trimethylate H3-K9.Wu, H. et al. (2010) PLoS One 5, e8570,doi:10.1371/journal.pone.0008570).

Human EZH2 nucleic acids and polypeptides have previously beendescribed. See, e.g., Chen et al. (1996) Genomics 38:30-7 [746 aminoacids]; Swiss-Prot Accession No. Q15910 [746 amino acids]; GenBankAccession Nos. NM_(—)004456 and NP_(—)004447 (isoform a [751 aminoacids]); and GenBank Accession Nos. NM_(—)152998 and NP_(—)694543(isoform b [707 amino acids]), each of which is incorporated herein byreference in its entirety.

Amino acid sequence of human EZH2 (Swiss-Prot Accession No. Q15910)(SEQ ID NO: 1)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPmRNA sequence of human EZH2, transcript variant 1(GenBank Accession No. NM_004456) (SEQ ID NO: 2)ggcggcgcttgattgggctgggggggccaaataaaagcgatggcgattgggctgccgcgtttggcgctcggtccggtcgcgtccgacacccggtgggactcagaaggcagtggagccccggcggcggcggcggcggcgcgcgggggcgacgcgcgggaacaacgcgagtcggcgcgcgggacgaagaataatcatgggccagactgggaagaaatctgagaagggaccagtttgttggcggaagcgtgtaaaatcagagtacatgcgactgagacagctcaagaggttcagacgagctgatgaagtaaagagtatgtttagttccaatcgtcagaaaattttggaaagaacggaaatcttaaaccaagaatggaaacagcgaaggatacagcctgtgcacatcctgacttctgtgagctcattgcgcgggactagggagtgttcggtgaccagtgacttggattttccaacacaagtcatcccattaaagactctgaatgcagttgcttcagtacccataatgtattcttggtctcccctacagcagaattttatggtggaagatgaaactgttttacataacattccttatatgggagatgaagttttagatcaggatggtactttcattgaagaactaataaaaaattatgatgggaaagtacacggggatagagaatgtgggtttataaatgatgaaatttttgtggagttggtgaatgcccttggtcaatataatgatgatgacgatgatgatgatggagacgatcctgaagaaagagaagaaaagcagaaagatctggaggatcaccgagatgataaagaaagccgcccacctcggaaatttccttctgataaaatttttgaagccatttcctcaatgtttccagataagggcacagcagaagaactaaaggaaaaatataaagaactcaccgaacagcagctcccaggcgcacttcctcctgaatgtacccccaacatagatggaccaaatgctaaatctgttcagagagagcaaagcttacactcctttcatacgcttttctgtaggcgatgttttaaatatgactgcttcctacatcgtaagtgcaattattcttttcatgcaacacccaacacttataagcggaagaacacagaaacagctctagacaacaaaccttgtggaccacagtgttaccagcatttggagggagcaaaggagtttgctgctgctctcaccgctgagcggataaagaccccaccaaaacgtccaggaggccgcagaagaggacggcttcccaataacagtagcaggcccagcacccccaccattaatgtgctggaatcaaaggatacagacagtgatagggaagcagggactgaaacggggggagagaacaatgataaagaagaagaagagaagaaagatgaaacttcgagctcctctgaagcaaattctcggtgtcaaacaccaataaagatgaagccaaatattgaacctcctgagaatgtggagtggagtggtgctgaagcctcaatgtttagagtcctcattggcacttactatgacaatttctgtgccattgctaggttaattgggaccaaaacatgtagacaggtgtatgagtttagagtcaaagaatctagcatcatagctccagctcccgctgaggatgtggatactcctccaaggaaaaagaagaggaaacaccggttgtgggctgcacactgcagaaagatacagctgaaaaaggacggctcctctaaccatgtttacaactatcaaccctgtgatcatccacggcagccttgtgacagttcgtgcccttgtgtgatagcacaaaatttttgtgaaaagttttgtcaatgtagttcagagtgtcaaaaccgctttccgggatgccgctgcaaagcacagtgcaacaccaagcagtgcccgtgctacctggctgtccgagagtgtgaccctgacctctgtcttacttgtggagccgctgaccattgggacagtaaaaatgtgtcctgcaagaactgcagtattcagcggggctccaaaaagcatctattgctggcaccatctgacgtggcaggctgggggatttttatcaaagatcctgtgcagaaaaatgaattcatctcagaatactgtggagagattatttctcaagatgaagctgacagaagagggaaagtgtatgataaatacatgtgcagctttctgttcaacttgaacaatgattttgtggtggatgcaacccgcaagggtaacaaaattcgttttgcaaatcattcggtaaatccaaactgctatgcaaaagttatgatggttaacggtgatcacaggataggtatttttgccaagagagccatccagactggcgaagagctgttttttgattacagatacagccaggctgatgccctgaagtatgtcggcatcgaaagagaaatggaaatcccttgacatctgctacctcctcccccctcctctgaaacagctgccttagcttcaggaacctcgagtactgtgggcaatttagaaaaagaacatgcagtttgaaattctgaatttgcaaagtactgtaagaataatttatagtaatgagtttaaaaatcaactttttattgccttctcaccagctgcaaagtgttttgtaccagtgaatttttgcaataatgcagtatggtacatttttcaactttgaataaagaatacttgaacttgtccttgttgaatcFull amino acid of EZH2, isoform a (GenBank Accession No. NP_004447)(SEQ ID NO: 3)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHRKCNYSFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPmRNA sequence of human EZH2, transcript variant 2(GenBank Accession No. NM_152998) (SEQ ID NO: 4)ggcggcgcttgattgggctgggggggccaaataaaagcgatggcgattgggctgccgcgtttggcgctcggtccggtcgcgtccgacacccggtgggactcagaaggcagtggagccccggcggcggcggcggcggcgcgcgggggcgacgcgcgggaacaacgcgagtcggcgcgcgggacgaagaataatcatgggccagactgggaagaaatctgagaagggaccagtttgttggcggaagcgtgtaaaatcagagtacatgcgactgagacagctcaagaggttcagacgagctgatgaagtaaagagtatgtttagttccaatcgtcagaaaattttggaaagaacggaaatcttaaaccaagaatggaaacagcgaaggatacagcctgtgcacatcctgacttctgtgagctcattgcgcgggactagggaggtggaagatgaaactgttttacataacattccttatatgggagatgaagttttagatcaggatggtactttcattgaagaactaataaaaaattatgatgggaaagtacacggggatagagaatgtgggtttataaatgatgaaatttttgtggagttggtgaatgcccttggtcaatataatgatgatgacgatgatgatgatggagacgatcctgaagaaagagaagaaaagcagaaagatctggaggatcaccgagatgataaagaaagccgcccaCctcggaaatttccttctgataaaatttttgaagccatttcctcaatgtttccagataagggcacagcagaagaactaaaggaaaaatataaagaactcaccgaacagcagctCccaggcgcacttcctcctgaatgtacccccaacatagatggaccaaatgctaaatctgttcagagagagcaaagcttacactcctttcatacgcttttctgtaggcgatgttttaaatatgactgcttcctacatccttttcatgcaacacccaacacttataagcggaagaacacagaaacagctctagacaacaaaccttgtggaccacagtgttaccagcatttggagggagcaaaggagtttgctgctgctctcaccgctgagcggataaagaccccaccaaaacgtccaggaggccgcagaagaggacggcttcccaataacagtagcaggcccagcacccccaccattaatgtgctggaatcaaaggatacagacagtgatagggaagcagggactgaaacggggggagagaacaatgataaagaagaagaagagaagaaagatgaaacttcgagctcctctgaagcaaattctcggtgtcaaacaccaataaagatgaagccaaatattgaacctcctgagaatgtggagtggagtggtgctgaagcctcaatgtttagagtcctcattggcacttactatgacaatttctgtgccattgctaggttaattgggaccaaaacatgtagacaggtgtatgagtttagagtcaaagaatctagcatcatagctccagctcccgctgaggatgtggatactcctccaaggaaaaagaagaggaaacaccggttgtgggctgcacactgcagaaagatacagctgaaaaaggacggctcctctaaccatgtttacaactatcaaccctgtgatcatccacggcagccttgtgacagttcgtgcccttgtgtgatagcacaaaatttttgtgaaaagttttgtcaatgtagttcagagtgtcaaaaccgctttccgggatgccgctgcaaagCaCagtgcaacaccaagcagtgcccgtgctacctggctgtccgagagtgtgaccctgaCctCtgtCttacttgtggagccgctgaccattgggacagtaaaaatgtgtcctgcaagaaCtgcagtattcagcggggctccaaaaagcatctattgctggcaccatctgacgtggcaggctgggggatttttatcaaagatcctgtgcagaaaaatgaattcatctcagaatactgtggagagattatttctcaagatgaagctgacagaagagggaaagtgtatgataaatacatgtgcagctttctgttcaacttgaacaatgattttgtggtggatgcaacccgcaagggtaacaaaattcgttttgcaaatcattcggtaaatccaaactgctatgcaaaagttatgatggttaacggtgatcacaggataggtatttttgccaagagagccatccagactggcgaagagctgttttttgattacagatacagccaggctgatgccctgaagtatgtcggcatcgaaagagaaatggaaatcccttgacatctgctacctcctcccccctcctctgaaacagctgccttagcttcaggaacctcgagtactgtgggcaatttagaaaaagaacatgcagtttgaaattctgaatttgcaaagtactgtaagaataatttatagtaatgagtttaaaaatcaactttttattgccttctcaccagctgcaaagtgttttgtaccagtgaatttttgcaataatgcagtatggtacatttttcaactttgaataaagaatacttgaacttgtc cttgttgaatcFull amino acid of EZH2, isoform b (GenBank Accession No. NP_694543)(SEQ ID NO: 5) MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTREVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGI EREMEIP

As mentioned above, the catalytic site of EZH2 is believed to reside ina conserved domain of the protein known as the SET domain. The aminoacid sequence of the SET domain of EZH2 is provided by the followingpartial sequence spanning amino acid residues 613-726 of Swiss-ProtAccession No. Q15910 (SEQ ID NO: 1):

(SEQ ID NO: 6)HLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDY.The tyrosine (Y) residue shown underlined in SEQ ID NO: 6 is Tyr641(Y641) in Swiss-Prot Accession No. Q15910 (SEQ ID NO: 1).

The SET domain of GenBank Accession No. NP_(—)004447 (SEQ ID NO: 3)spans amino acid residues 618-731 and is identical to SEQ ID NO:6. Thetyrosine residue corresponding to Y641 in Swiss-Prot Accession No.Q15910 shown underlined in SEQ ID NO: 6 is Tyr646 (Y646) in GenBankAccession No. NP_(—)004447 (SEQ ID NO: 3).

The SET domain of GenBank Accession No. NP_(—)694543 (SEQ ID NO: 5)spans amino acid residues 574-687 and is identical to SEQ ID NO: 6. Thetyrosine residue corresponding to Y641 in Swiss-Prot Accession No.Q15910 shown underlined in SEQ ID NO: 6 is Tyr602 (Y602) in GenBankAccession No. NP_(—)694543 (SEQ ID NO: 5).

The nucleotide sequence encoding the SET domain of GenBank Accession No.NP_(—)004447 is

(SEQ ID NO: 7)catctattgctggcaccatctgacgtggcaggctgggggatttttatcaaagatcctgtgcagaaaaatgaattcatctcagaatactgtggagagattatttctcaagatgaagctgacagaagagggaaagtgtatgataaatacatgtgcagctttctgttcaacttgaacaatgattttgtggtggatgcaacccgcaagggtaacaaaattcgttttgcaaatcattcggtaaatccaaactgctatgcaaaagttatgatggttaacggtgatcacaggataggtatttttgccaagagagccatccagactggcgaagagctgttttttgattac,where the codon encoding Y641 is shown underlined.

For purposes of this application, amino acid residue Y641 of human EZH2is to be understood to refer to the tyrosine residue that is orcorresponds to Y641 in Swiss-Prot Accession No. Q15910.

Full amino acid sequence of Y641 mutant EZH2 (SEQ ID NO: 8)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEXCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPWherein x can be any amino acid residue other than tyrosine (Y)

Also for purposes of this application, a Y641 mutant of human EZH2, and,equivalently, a Y641 mutant of EZH2, is to be understood to refer to ahuman EZH2 in which the amino acid residue corresponding to Y641 ofwild-type human EZH2 is substituted by an amino acid residue other thantyrosine.

In one embodiment the amino acid sequence of a Y641 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of a single amino acid residue corresponding to Y641 ofwild-type human EZH2 by an amino acid residue other than tyrosine.

In one embodiment the amino acid sequence of a Y641 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of phenylalanine (F) for the single amino acid residuecorresponding to Y641 of wild-type human EZH2. The Y641 mutant of EZH2according to this embodiment is referred to herein as a Y641F mutant or,equivalently, Y641F.

Y641F (SEQ ID NO: 9)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEFCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIP

In one embodiment the amino acid sequence of a Y641 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of histidine (H) for the single amino acid residuecorresponding to Y641 of wild-type human EZH2. The Y641 mutant of EZH2according to this embodiment is referred to herein as a Y641H mutant or,equivalently, Y641H.

Y641H (SEQ ID NO: 10)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGTINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEHCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIP

In one embodiment the amino acid sequence of a Y641 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of asparagine (N) for the single amino acid residuecorresponding to Y641 of wild-type human EZH2. The Y641 mutant of EZH2according to this embodiment is referred to herein as a Y641N mutant or,equivalently, Y641N.

Y641N (SEQ ID NO: 11)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRERRADEVKSMESSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNECAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISENCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIP

In one embodiment the amino acid sequence of a Y641 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of serine (S) for the single amino acid residuecorresponding to Y641 of wild-type human EZH2. The Y641 mutant of EZH2according to this embodiment is referred to herein as a Y641S mutant or,equivalently, Y641S.

Y641S (SEQ ID NO: 12)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRERRADEVKSMESSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKEPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISESCGEIISQDEADRRGKVYDKYMCSFLFNLNNDEVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIP

The tolerance for multiple Y641 mutations in EZH2 suggests that arelease of steric crowding may allow greater access for proper alignmentof the larger dimethyl lysine as the substrate for thedi-to-trimethylation reaction. Crystallographic analysis of the proteinmethyltransferases SET7/9 and G9a reveals that the side chain hydroxylsof the active site tyrosine residues are involved in H-bondinginteractions directly with the amine of the methyl-accepting lysine, orindirectly through an intervening water molecule. While the largeractive site of the Y641 mutants is favorable for di- andtri-methylation, the loss of the tyrosine hydroxyl hydrogen bondacceptor may result in an unfavorable orientation of the active site forinitial methyl transfer to the lysine amine.

The implications of the present results for human disease are made clearby the data summarized in Table 1 (see below). Cells heterozygous forEZH2 would be expected to display a malignant phenotype due to theefficient formation of H3-K27me1 by the WT enzyme and the efficient,subsequent transition of this progenitor species to H3-K27me2, and,especially, H3-K27me3, by the mutant enzyme form(s).

It has been reported that H3-K27me1 formation is not exclusivelydependent on WT-EZH2 catalysis. Knockout studies of EZH2 and of anotherPRC2 subunit, EED, have demonstrated H3-K27me1 formation can becatalyzed by PRC2 complexes containing either EZH2 or the relatedprotein EZH1 as the catalytic subunit. Shen, X. et al. (2008) Mol Cell32:491-502. Hence, catalytic coupling between the mutant EZH2 speciesand PRC2 complexes containing either WT-EZH2 or WT-EZH1 would suffice toaugment H3-K27me2/3 formation, and thus produce the attendant malignantphenotype. The data therefore suggest that the malignant phenotype offollicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) ofthe germinal center B cell (GCB) subtype, associated with expression ofmutant forms of EZH2, is the result of an overall gain of function withrespect to formation of the trimethylated form of H3-K27. Thisinterpretation of the data also helps to reconcile the existence ofcancer-associated overexpression of EZH2 or PRC2 associated proteins(e.g., PHF19/PCL3) and also loss-of-function genotypes for the histoneH3-K27 demethylase UTX. Loss of UTX activity would be enzymaticallyequivalent to a gain of function for EZH2, in either situation resultingin greater steady state levels of tri-methylated H3-K27 in cancer cells(FIG. 4).

The mono-, di-, and tri- methylation states of histone H3-K27 areassociated with different functions in transcriptional control. HistoneH3-K27 monomethylation is associated with active transcription of genesthat are poised for transcription. Cui et al. (2009) Cell Stem Cell4:80-93; Barski (2007) Cell 129:823-37. In contrast, trimethylation ofhistone H3-K27 is associated with either transcriptionally repressedgenes or genes that are poised for transcription when histone H3-K4trimethylation is in cis. Cui et al. (supra); Kirmizis et al. (2007)Genes Dev 18:1592-1605; Bernstein et al. (2006) Cell 125:315-26. Takentogether, alterations in the PRC2 complex activity reported in cancer,including the Y641 mutation of EZH2, are predicted to result in anincrease in the trimethylated state of histone H3-K27 and thus to resultin transcriptional repression.

Another discovery of the present invention is that cells expressing Y641mutant EZH2 are, in general, more sensitive to small molecule EZH2inhibitors than cells expressing WT EZH2. Specifically, cells expressingY641 mutant EZH2 show reduced growing, dividing or proliferation, oreven undergo apoptosis or necrosis after the treatment of EZH2inhibitors. In contrast, cells expressing WT EZH2 are not responsive tothe anti-proliferative effect of the EZH2 inhibitors (FIGS. 13 and 14).Another surprising discovery of the present invention is that it ispossible for cells expressing WT EZH2 to display a similar methylationstatus of histone H3-K27 as cells expressing Y641 EZH2, and that thismethylation status can also correlate with sensitivity to an EZH2inhibitor independently of EZH2 mutational status. In general, globalH3-K27me3 levels are similar or higher in Y641 mutant containing celllines than in cell lines expressing WT EZH2; however levels of H3-K27me2are dramatically lower in EZH2 Y641 mutant cell lines and certain wildtype cell lines, such as the Pfeiffer cell line than in other wild typecell lines (FIGS. 9, 10 and 11). Thus the ratio of H3-K27me2/me3 signalin Y641 mutant lines and the Pfeiffer cell line is much lower than thatobserved in other WT lines. The present data further demonstrate thatcell lines with low H3-K27me2 signal and similar or higher H3-K27me3signal relative to typical WT EZH2 expressing cells lines are moresensitive to small molecule EZH2 inhibitors. Specifically, cells with alowH3-K27me2 signal and a normal or high H3K27me3 signal stop dividingor even die after treatment with EZH2 inhibitors (FIGS. 9, 10, 11, 13,and 14). In contrast, cells with a higher ratio of H3-K27me2/me3 signalare not responsive to the anti-proliferative effect of the EZH2inhibitors (FIGS. 9, 10, 11, 13, and 14). The instant invention providespreviously unknown and unexpected results that identifying EZH2 Y641mutations in patient tumors and/or detecting low levels of H3-K27me2 andnormal or high levels of H3-K27me3 relative to a control, through use oftechniques such as western blot, MS or IHC in a patient can be used toidentify which patient will respond to an EZH2 inhibitor treatment.

EZH2 and other protein methyltransferases have been suggested to beattractive targets for drug discovery. Copeland et al. (2009) Nat RevDrug Discov 8:724-32; Copeland et al. (2010) Curr Opin Chem Biol14(4):505-10; Pollock et al. (2010) Drug Discovery Today: TherapeuticStrategies 6(1):71-9. The present data also suggest an experimentalstrategy for development of FL and GCB lymphoma-specific drugs. As thedifferences in substrate recognition between the WT anddisease-associated mutants derive from transition state interactions,small molecule inhibitors that selectively mimic the transition state ofthe mutant EZH2 over that of the WT enzyme should prove to be effectivein blocking H3-K27 methylation in mutation-bearing cells. Inhibitors ofthis type would be expected to display a large therapeutic index, astarget-mediated toxicity would be minimal for any cells bearing only theWT enzyme. Transition state mimicry has proved to be an effectivestrategy for drug design in many disease areas. See, for example,Copeland, R. A. Enzymes: A Practical Introduction to Structure,Mechanism and Data Analysis. 2nd ed, (Wiley, 2000).

The present results point to a previously unrecognized, surprisingdependency on enzymatic coupling between enzymes that perform H3-K27mono-methylation and certain mutant forms of EZH2 for pathogenesis infollicular lymphoma and diffuse large B-cell lymphoma. While notintending to be bound by any one theory, it is believed the dataconstitute the first example of a human disease that is dependent onsuch coupling of catalytic activity between normal (WT) anddisease-associated mutant (Y641) enzymes.

An aspect of the invention is a method for inhibiting in a subjectconversion of H3-K27 to trimethylated H3-K27. The inhibition can involveinhibiting in a subject conversion of unmethylated H3-K27 tomonomethylated H3-K27, conversion of monomethylated H3-K27 todimethylated H3-K27, conversion of dimethylated H3-K27 to trimethylatedH3-K27, or any combination thereof, including, for example, conversionof monomethylated H3-K27 to dimethylated H3-K27 and conversion ofdimethylated H3-K27 to trimethylated H3-K27. As used herein,unmethylated H3-K27 refers to histone H3 with no methyl group covalentlylinked to the amino group of lysine 27. As used herein, monomethylatedH3-K27 refers to histone H3 with a single methyl group covalently linkedto the amino group of lysine 27. Monomethylated H3-K27 is also referredto herein as H3-K27me1. As used herein, dimethylated H3-K27 refers tohistone H3 with two methyl groups covalently linked to the amino groupof lysine 27. Dimethylated H3-K27 is also referred to herein asH3-K27me2. As used herein, trimethylated H3-K27 refers to histone H3with three methyl groups covalently linked to the amino group of lysine27. Trimethylated H3-K27 is also referred to herein as H3-K27me3.

Histone H3 is a 136 amino acid long protein, the sequence of which isknown. See, for example, GenBank Accession No. CAB02546, the contents ofwhich is incorporated herein by reference. As disclosed further herein,in addition to full-length histone H3, peptide fragments of histone H3comprising the lysine residue corresponding to K27 of full-lengthhistone H3 can be used as substrate for EZH2 (and likewise for mutantforms of EZH2) to assess conversion of H3-K27 ml to H3-K27 m2 andconversion of H3-K27 m2 to H3-K27 m3. In one embodiment, such peptidefragment corresponds to amino acid residues 21-44 of histone H3. Suchpeptide fragment has the amino acid sequence LATKAARKSAPATGGVKKPHRYRP(SEQ ID NO: 13).

The method involves administering to a subject expressing a Y641 mutantof EZH2 a therapeutically effective amount of an inhibitor of EZH2,wherein the inhibitor inhibits histone methyltransferase activity ofEZH2, thereby inhibiting conversion of H3-K27 to trimethylated H3-K27 inthe subject. In one embodiment a subject expressing a Y641 mutant ofEZH2 refers to a subject having a detectable amount of a Y641 mutantEZH2 polypeptide. In one embodiment a subject expressing a Y641 mutantof EZH2 refers to a subject having a detectable amount of a nucleic acidencoding a Y641 mutant EZH2 polypeptide.

A Y641 mutant EZH2 polypeptide can be detected using any suitablemethod. For example, a Y641 mutant EZH2 polypeptide can be detectedusing an antibody that binds specifically to the Y641 mutant EZH2polypeptide or to a peptide fragment that is characteristic of the Y641mutant EZH2 polypeptide. A peptide fragment that is characteristic ofthe Y641 mutant EZH2 polypeptide may include, for example, a SET domainas provided in SEQ ID NO: 6, except for substitution of Y641 by an aminoacid residue other than tyrosine. In another embodiment, a peptidefragment that is characteristic of the Y641 mutant EZH2 polypeptide mayinclude, for example, a 10-113 amino acid fragment of the SET domain asprovided in SEQ ID NO: 6, except for substitution of Y641 by an aminoacid residue other than tyrosine, provided that the fragment includesthe amino acid residue corresponding to Y641. It is expected that theepitope for such antibody includes the amino acid residue correspondingto Y641 of wild-type EZH2. An antibody is considered to bindspecifically to the Y641 mutant EZH2 polypeptide or to a peptidefragment that is characteristic of the Y641 mutant EZH2 polypeptide ifit binds to that mutant EZH2 polypeptide or peptide fragment thereof butnot to the corresponding wild-type EZH2 polypeptide or peptide fragmentthereof. In one embodiment, such antibody is considered to bindspecifically to the Y641 mutant EZH2 polypeptide or to a peptidefragment that is characteristic of the Y641 mutant EZH2 polypeptide ifit binds to that mutant EZH2 polypeptide or peptide fragment thereofwith an affinity that is at least ca. 100-fold greater than for thecorresponding wild-type EZH2 polypeptide or peptide fragment thereof. Inone embodiment, such antibody is considered to bind specifically to theY641 mutant EZH2 polypeptide or to a peptide fragment that ischaracteristic of the Y641 mutant EZH2 polypeptide if it binds to thatmutant EZH2 polypeptide or peptide fragment thereof with an affinitythat is at least ca. 1000-fold greater than for the correspondingwild-type EZH2 polypeptide or peptide fragment thereof. The antibody canbe used, for example, in an enzyme-linked immunosorbent assay (ELISA) orWestern blot assay.

In one embodiment the antibody is a monoclonal antibody. A monoclonalantibody can be prepared according to conventional methods well known inthe art. See, for example, Köhler and Milstein (1975) Nature 256(5517):495-7.

As another example, a Y641 mutant EZH2 polypeptide can be detected usingmass spectrometry (MS), e.g., electrospray ionization coupled withtime-of-flight (ESI-TOF) or matrix-assisted laser desorption/ionizationcoupled with time-of-flight (MALDI-TOF). Such methods are well known inthe art. The analysis will involve identification of one or more peptidefragments comprising the mutation of interest, for example, a peptide 12to 24 amino acids long comprising a sequence spanning the amino acidcorresponding to Y641 in wild-type EZH2.

A nucleic acid encoding a Y641 mutant EZH2 polypeptide or a peptidefragment that is characteristic of the Y641 mutant EZH2 polypeptide canbe detected using any suitable method. For example, a nucleic acidencoding a Y641 mutant EZH2 polypeptide can be detected usingwhole-genome resequencing or target region resequencing (the latter alsoknown as targeted resequencing) using suitably selected sources of DNAand polymerase chain reaction (PCR) primers in accordance with methodswell known in the art. See, for example, Bentley (2006) Curr Opin GenetDev. 16:545-52, and Li et al. (2009) Genome Res 19:1124-32. The methodtypically and generally entails the steps of genomic DNA purification,PCR amplification to amplify the region of interest, cycle sequencing,sequencing reaction cleanup, capillary electrophoresis, and dataanalysis. High quality PCR primers to cover region of interest aredesigned using in silico primer design tools. Cycle sequencing is asimple method in which successive rounds of denaturation, annealing, andextension in a thermal cycler result in linear amplification ofextension products. The products are typically terminated with afluorescent tag that identifies the terminal nucleotide base as G, A, T,or C. Unincorporated dye terminators and salts that may compete forcapillary eletrophoretic injection are removed by washing. Duringcapillary electrophoresis, the products of the cycle sequencing reactionmigrate through capillaries filled with polymer. The negatively chargedDNA fragments are separated by size as they move through the capillariestoward the positive electrode. After electrophoresis, data collectionsoftware creates a sample file of the raw data. Using downstreamsoftware applications, further data analysis is performed to translatethe collected color data images into the corresponding nucleotide bases.Alternatively or in addition, the method may include the use ofmicroarray-based targeted region genomic DNA capture and/or sequencing.Kits, reagents, and methods for selecting appropriate PCR primers andperforming resequencing are commercially available, for example, fromApplied Biosystems, Agilent, and NimbleGen (Roche Diagnostics GmbH).Methods such as these have been used to detect JAK2 andmyeloproliferative leukemia gene (MPL) mutations and to diagnosepolycythemia vera, essential thrombocythemia, and idiopathicmyelofibrosis. For use in the instant invention, PCR primers may beselected so as to amplify, for example, at least a relevant portion ofSEQ ID NO: 7 (above).

Alternatively or in addition, a nucleic acid encoding a Y641 mutant EZH2polypeptide may be detected using a Southern blot in accordance withmethods well known in the art. In one embodiment a DNA sequence encodinga Y641 mutant EZH2 polypeptide is detected using nucleic acidhybridization performed under highly stringent conditions. A nucleicacid probe is selected such that its sequence is complementary to atarget nucleic acid sequence that includes a codon for the mutant aminoacid corresponding to Y641 of wild-type EZH2.

A sequence-specific probe is combined with a sample to be tested underhighly stringent conditions. The term “highly stringent conditions” asused herein refers to parameters with which the art is familiar. Nucleicacid hybridization parameters may be found in references that compilesuch methods, e.g., J. Sambrook, et al., eds., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989, or F. M. Ausubel, et al., eds., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., New York. Morespecifically, highly stringent conditions, as used herein, refers, forexample, to hybridization at 65° C. in hybridization buffer (3.5×SSC,0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% bovine serum albumin(BSA), 2.5 mM NaH₂PO₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodiumchloride/0.015 M sodium citrate, pH 7; SDS is sodium dodecyl sulphate;and EDTA is ethylenediaminetetracetic acid. After hybridization, themembrane upon which the DNA is transferred is washed, for example, in2×SSC at room temperature and then at 0.1-0.5×SSC/0.1×SDS attemperatures up to 68° C.

There are other conditions, reagents, and so forth that can be used,which result in a similar degree of stringency. The skilled artisan willbe familiar with such conditions, and thus they are not given here. Itwill be understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof EZH2-associated nucleic acids of the invention, including, inparticular, nucleic acids encoding Y641 mutants of EZH2 (e.g., by usinglower stringency conditions). The skilled artisan also is familiar withthe methodology for screening cells and libraries for expression of suchmolecules, which then are routinely isolated, followed by isolation ofthe pertinent nucleic acid molecule and sequencing.

The subject is administered a therapeutically effective amount of aninhibitor of EZH2. As used herein, an inhibitor of EZH2 refers,generally, to a small molecule, i.e., a molecule of molecular weightless than about 1.5 kilodaltons (kDa), which is capable of interferingwith the histone methyltransferase enzymatic activity of EZH2.

In one embodiment the inhibitor of EZH2 inhibits histonemethyltransferase activity of wild-type EZH2. In one embodiment theinhibitor of EZH2 inhibits histone methyltransferase activity of theY641 mutant of EZH2. In one embodiment the inhibitor of EZH2 inhibitshistone methyltransferase activity of wild-type EZH2 and histonemethyltransferase activity of the Y641 mutant of EZH2. In one embodimentthe inhibitor of EZH2 selectively inhibits histone methyltransferaseactivity of the Y641 mutant of EZH2.

As disclosed herein, certain Y641 mutants of EZH2 are relatively poorcatalysts for conversion of unmethylated H3-K27 to H3-K27me1 and yetunexpectedly effective catalysts for conversion of H3-K27me2 toH3-K27me3. Conversely, wild-type EZH2 is a relatively effective catalystfor conversion of unmethylated H3-K27 to H3-K27me1 and yet unexpectedlyineffective catalyst for conversion of H3-K27me2 to H3-K27me3. This isimportant because mono-, di- and tri-methylated states of H3-K27 exhibitdifferent functions in transcriptional control. For example, H3-K27me1is associated with active transcription of genes that are poised fortranscription, while H3-K27me3 is associated with transcriptionallyrepressed genes or genes that are poised for transcription when H3-K4trimethylation is in cis. Thus, selective inhibition of histonemethyltransferase activity of the Y641 mutant of EZH2 effects selectiveinhibition of production of the trimethylated form of H3-K27, therebyfavoring transcription associated with H3-K27me1 and disfavoringrepression of transcription associated with H3-K27me3.

An inhibitor of EZH2 “selectively inhibits” histone methyltransferaseactivity of the Y641 mutant of EZH2 when it inhibits histonemethyltransferase activity of the Y641 mutant of EZH2 more effectivelythan it inhibits histone methyltransferase activity of wild-type EZH2.For example, in one embodiment the selective inhibitor has an IC50 forthe Y641 mutant of EZH2 that is at least 40 percent lower than the IC50for wild-type EZH2. In one embodiment the selective inhibitor has anIC50 for the Y641 mutant of EZH2 that is at least 50 percent lower thanthe IC50 for wild-type EZH2. In one embodiment the selective inhibitorhas an IC50 for the Y641 mutant of EZH2 that is at least 60 percentlower than the IC50 for wild-type EZH2. In one embodiment the selectiveinhibitor has an IC50 for the Y641 mutant of EZH2 that is at least 70percent lower than the IC50 for wild-type EZH2. In one embodiment theselective inhibitor has an IC50 for the Y641 mutant of EZH2 that is atleast 80 percent lower than the IC50 for wild-type EZH2. In oneembodiment the selective inhibitor has an IC50 for the Y641 mutant ofEZH2 that is at least 90 percent lower than the IC50 for wild-type EZH2.

In one embodiment, the selective inhibitor of a Y641 mutant of EZH2exerts essentially no inhibitory effect on wild-type EZH2.

The inhibitor inhibits conversion of H3-K27me2 to H3-K27me3. In oneembodiment the inhibitor is said to inhibit trimethylation of H3-K27.Since conversion of H3-K27me1 to H3-K27me2 precedes conversion ofH3-K27me2 to H3-K27me3, an inhibitor of conversion of H3-K27me1 toH3-K27me2 naturally also inhibits conversion of H3-K27me2 to H3-K27me3,i.e., it inhibits trimethylation of H3-K27. It is also possible toinhibit conversion of H3-K27me2 to H3-K27me3 without inhibition ofconversion of H3-K27me1 to H3-K27me2. Inhibition of this type would alsoresult in inhibition of trimethylation of H3-K27, albeit withoutinhibition of dimethylation of H3-K27.

In one embodiment the inhibitor inhibits conversion of H3-K27me1 toH3-K27me2 and the conversion of H3-K27me2 to H3-K27me3. Such inhibitormay directly inhibit the conversion of H3-K27me1 to H3-K27me2 alone.Alternatively, such inhibitor may directly inhibit both the conversionof H3-K27me1 to H3-K27me2 and the conversion of H3-K27me2 to H3-K27me3.

The inhibitor inhibits histone methylase activity. Inhibition of histonemethylase activity can be detected using any suitable method. Theinhibition can be measured, for example, either in terms of rate ofhistone methylase activity or as product of histone methylase activity.Methods suitable for either of these readouts are included in theExamples below.

The inhibition is a measurable inhibition compared to a suitablenegative control. In one embodiment, inhibition is at least 10 percentinhibition compared to a suitable negative control. That is, the rate ofenzymatic activity or the amount of product with the inhibitor is lessthan or equal to 90 percent of the corresponding rate or amount madewithout the inhibitor. In various other embodiments, inhibition is atleast 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, or 95 percent inhibitioncompared to a suitable negative control. In one embodiment, inhibitionis at least 99 percent inhibition compared to a suitable negativecontrol. That is, the rate of enzymatic activity or the amount ofproduct with the inhibitor is less than or equal to 1 percent of thecorresponding rate or amount made without the inhibitor.

In one embodiment, the inhibitor is S-adenosyl-L-homocysteine (SAH). SAHhas the structural formula

and is commercially available from a number of suppliers, including, forexample, Sigma-Aldrich, St. Louis, Mo. SAH has been described as aninhibitor of transmethylation by 5-adenosylmethionine-dependentmethyltransferases.

In one embodiment, the inhibitor is Compound 75

or a pharmaceutically acceptable salt thereof.

In certain embodiments the invention comprises the step of performing anassay to detect a Y641 mutant of EZH2 in a sample from a subject. Assaysof this type are described above. As used herein, a “sample from asubject” refers to any suitable sample containing cells or components ofcells obtained or derived from a subject. In one embodiment the sampleincludes cells suspected to express Y641 mutant of EZH2, e.g., cancercells. In one embodiment the sample is a blood sample. In one embodimentthe sample is a biopsy sample obtained from, for example, a lymphatictissue (e.g., lymph node) or bone marrow. In one embodiment the sampleis a biopsy sample obtained from a tissue other than or in addition to alymphatic tissue (e.g., lymph node) or bone marrow. For example, in oneembodiment the sample is a biopsy from a cancer, e.g., a tumor composedof cancer cells. Cells in the sample can be isolated from othercomponents of the sample. For example, peripheral blood mononuclearcells (PBMCs) can be isolated as a buffy coat from a blood sample thathas been centrifuged in accordance with methods familiar to those ofskill in the art.

When the result of the assay on a sample from a subject indicates that aY641 mutant of EZH2 is present in the sample, the subject is said toexpress the Y641 mutant of EZH2. Indeed, in one embodiment, when theresult of the assay on a sample from a subject indicates that a Y641mutant of EZH2 is present in the sample, the subject is identified as acandidate for treatment with an inhibitor of EZH2, wherein the inhibitorselectively inhibits histone methyltransferase activity of the Y641mutant of EZH2.

When the result of the assay on a sample from a cancer indicates that aY641 mutant of EZH2 is present in the cancer, the cancer is said toexpress the Y641 mutant of EZH2.

Similarly, when the result of the assay on a sample comprising cancercells from a subject having a cancer indicates that a Y641 mutant ofEZH2 is present in the sample, the subject is said to express the Y641mutant of EZH2.

The present invention also provides a previously unrecognized,surprising correlation of a patient's responsiveness to an EZH2inhibitor with the H3-K27me2 level or preferably with the levels ofH3-K27me and H3-K27me3. For example, cells with a low H3-K27me2 andnormal or high me3 levels relative to a control are much more responsiveto the anti-proliferative effect of an EZH2 inhibitor than cells with anormal H3-K27 me2 and me3 levels.

An aspect of the invention is a method for determining responsiveness toan EZH2 inhibitor in a subject. In one embodiment the method includesisolating a tissue sample from the subject; detecting a dimethylation(me2) level of H3-K27 in the tissue sample; comparing the dimethylation(me2) level to a control dimethylation (me2) level; and identifying thesubject is responsive to said EZH2 inhibitor when the dimethylation(me2) level is absent or lower than the control dimethylation (me2)level. In one embodiment, the method further includes detecting atrimethylation (me3) level of H3-K27 in the tissue sample; comparing thetrimethylation (me3) level to a control trimethylation (me3) level andthe dimethylation (me2) level to a control dimethylation (me2) level;and identifying said subject is responsive to the EZH2 inhibitor whenthe trimethylation (me3) level is same as or higher than the controltrimethylation (me3) level and the dimethylation (me2) level is absentor lower than the control dimethylation (me2) level. In anotherembodiment, the method further includes obtaining a ratio of thedimethylation (me2) level to the trimethylation (me3) level of H3-K27 inthe tissue sample; obtaining a control ratio of the controldimethylation (me2) level to the control trimethylation (me3) level;comparing the ratio to the control ratio; and identifying the subject isresponsive to said EZH2 inhibitor when said ratio is lower than saidcontrol ratio. In a preferred embodiment, the subject has cancer. In oneembodiment, the cancer is a follicular lymphoma. Alternatively, thecancer is a diffuse large B-cell lymphoma (DLBCL). In another preferredembodiment, the subject expresses a Y641 mutant EZH2. In a preferredembodiment, the Y641 mutant is Y641F, Y641H, Y641N or Y641S.

Detection of dimethylated H3-K27 or trimethlated H3-K27 can beaccomplished using any suitable method in the art. In one embodiment,the methylation level is detected using antibodies specific fordimethylated H3-K27 or trimethlated H3-K27. For example, the isolatedtissue is formalin fixed and embedded in paraffin blocks for long termpreservation. The blocks can be used to prepare slides forimmunohistochemical staining or fluorescent staining with antibodiesagainst methylated H3-K27. Alternatively, whole cell lysates or histoneextracts can be prepared from the isolated tissue sample andsubsequently used for immunohistochemical staining, western blotanalysis or fluorescent staining. In another embodiment the methylationlevel is detected using a polypeptide or an aptamer specific fordimethylated H3-K27 or trimethlated H3-K27. In another embodiment, themethylation level is detected using mass spectrometry (MS).

A control dimethylated H3-K27 or a control trimethlated H3-K27 can beestablished from a control sample, e.g., an adjacent non-tumor tissueisolated from the subject or a healthy tissue from a healthy subject.Alternatively, the control methylation level of H3-K27me2 or H3-K27me3can be established by a pathologist with known methods in the art.

Screening Methods

An aspect of the invention is a method for identifying a test compoundas an inhibitor of a Y641 mutant of EZH2. In one embodiment the methodincludes combining an isolated Y641 mutant of EZH2 with a histonesubstrate, a methyl group donor (such as S-adenosyl methionine (SAM)),and a test compound, wherein the histone substrate comprises a form ofH3-K27 selected from the group consisting of unmethylated H3-K27,monomethylated H3-K27, dimethylated H3-K27, and any combination thereof;and performing an assay to detect methylation of H3-K27 in the histonesubstrate, thereby identifying the test compound as an inhibitor of theY641 mutant of EZH2 when methylation of H3-K27 in the presence of thetest compound is less than methylation of H3-K27 in the absence of thetest compound. The assay to detect methylation of H3-K27 can be selectedto measure the rate of methylation, the extent of methylation, or boththe rate and extent of methylation.

The Y641 mutant of EZH2 is isolated as a PRC2 complex or functionalequivalent thereof. As used herein, the term “isolated” meanssubstantially separated from other components with which the complex maybe found as it occurs in nature. A compound can be isolated withoutnecessarily being purified. In one embodiment the mutant of EZH2 isisolated as a complex of a Y641 mutant of EZH2 together with EED andSUZ12. In another embodiment the mutant of EZH2 is isolated as a complexof a Y641 mutant of EZH2 together with EED, SUZ12, and RbAp48. Underappropriate conditions, a PRC2 complex or functional equivalent thereofexhibits histone methyltransferase activity for H3-K27. In oneembodiment the complex is composed of recombinantly expressed componentpolypeptides, e.g., EZH2, EED, SUZ12, with or without RbAp48.

The isolated Y641 mutant of EZH2 is combined with a histone substrate. Ahistone substrate includes any suitable source of histone polypeptidesor fragments thereof that can serve as substrate for EZH2. In oneembodiment the histone substrate includes histones isolated from asubject. The histones can be isolated from cells of a subject using anysuitable method; such methods are well known to persons skilled in theart and need not be further specified here. See, for example, Fang etal. (2004) Methods Enzymol 377:213-26. In accordance with the Examplesbelow, in one embodiment the histone substrate is provided asnucleosomes. In accordance with the Examples below, in one embodimentthe histone substrate is provided as avian (chicken) erythrocytenucleosomes.

Histone substrate so provided may include an admixture of states ofhistone modification, including various states of H3-K27 methylation asjudged by Western blotting with H3-K27 methylation state-specificantibodies. In one embodiment the histone substrate may be provided aspurified full-length histone H3. Such purified full-length histone H3may be provided as a homogeneous preparation in respect of states ofH3-K27 methylation, or as an admixture of various states of H3-K27methylation. Homogeneous preparations of isolated histone H3 in respectof states of H3-K27 methylation may be prepared in part by passage overan immunoaffinity column loaded with suitable H3-K27 methylationstate-specific antibodies or by immunoprecipitation using magnetic beadscoated with suitable H3-K27 methylation state-specific antibodies.Alternatively or in addition, the methylation state of H3-K27 can becharacterized as part of performing the assay. For example, the startingmaterial histone substrate might be characterized as containing 50percent unmethylated H3-K27, 40 percent monomethylated H3-K27, 10percent dimethylated H3-K27, and 0 percent trimethylated H3-K27.

In one embodiment the histone substrate includes a peptide library or asuitable peptide comprising one or more amino acid sequences related tohistone H3, including, in particular, a sequence that encompassesH3-K27. For example, in one embodiment, the histone substrate is apeptide fragment that corresponds to amino acid residues 21-44 ofhistone H3. Such peptide fragment has the amino acid sequenceLATKAARKSAPATGGVKKPHRYRP (SEQ ID NO: 13). The peptide library or peptidecan be prepared by peptide synthesis according to techniques well knownin the art and optionally modified so as to incorporate any desireddegree of methylation of lysine corresponding to H3-K27. As described inthe Examples below, such peptides can also be modified to incorporate alabel, such as biotin, useful in performing downstream assays. In oneembodiment the label is appended to the amino (N)-terminus of thepeptide(s). In one embodiment the label is appended to the carboxy(C)-terminus of the peptide(s).

H3-K27 methylation-specific antibodies are available from a variety ofcommercial sources, including, for example, Cell Signaling Technology(Danvers, Mass.) and Active Motif (Carlsbad, Calif.).

The isolated Y641 mutant of EZH2 is combined with a test compound. Asused herein, a “test compound” refers to a small organic molecule havinga molecular weight of less than about 1.5 kDa. In one embodiment a testcompound is a known compound. In one embodiment a test compound is anovel compound. In one embodiment, a test compound can be provided aspart of a library of such compounds, wherein the library includes, forexample, tens, hundreds, thousands, or even more compounds. A library ofcompounds may advantageously be screened in a high throughput screeningassay, for example, using arrays of test compounds and roboticmanipulation in accordance with general techniques well known in theart.

In certain embodiments a test compound is a compound that is aderivative of SAH or a derivative of Compound 75.

Detection of methylation of H3-K27 can be accomplished using anysuitable method. In one embodiment, the source of donor methyl groupsincludes methyl groups that are labeled with a detectable label. Thedetectable label in one embodiment is an isotopic label, e.g., tritium.Other types of labels may include, for example, fluorescent labels.

Detection of formation of trimethylated H3-K27 can be accomplished usingany suitable method. For example, detection of formation oftrimethylated H3-K27 can be accomplished using an assay to detectincorporation of labeled methyl groups, such as described above,optionally combined with a chromatographic or other method to separatelabeled products by size, e.g., polyacrylamide gel electrophoresis(PAGE), capillary electrophoresis (CE), or high pressure liquidchromatography (HPLC). Alternatively or in addition, detection offormation of trimethylated H3-K27 can be accomplished using antibodiesthat are specific for trimethylated H3-K27.

Detection of conversion of monomethylated H3-K27 to dimethylated H3-K27can be accomplished using any suitable method. In one embodiment theconversion is measured using antibodies specific for monomethylatedH3-K27 and dimethylated H3-K27. For example, starting amounts orconcentrations of monomethylated H3-K27 and dimethylated H3-K27 may bedetermined using appropriate antibodies specific for monomethylatedH3-K27 and dimethylated H3-K27. Following the combination of enzyme,substrate, methyl group donor, and test compound, resulting amounts orconcentrations of monomethylated H3-K27 and dimethylated H3-K27 may thenbe determined using appropriate antibodies specific for monomethylatedH3-K27 and dimethylated H3-K27. The beginning and resulting amounts orconcentrations of monomethylated H3-K27 and dimethylated H3-K27 can thenbe compared. Alternatively or in addition, beginning and resultingamounts or concentrations of monomethylated H3-K27 and dimethylatedH3-K27 can then be compared to corresponding amounts of concentrationsfrom a negative control. A negative control reaction, in which no testagent is included in the assay, can be run in parallel or as ahistorical control. Results of such control reaction can optionally besubtracted from corresponding results of the experimental reaction priorto or in conjunction with making the comparison mentioned above.

Because the dimethylated form of H3-K27 may be further methylated in thesame assay, a reduction in the amount or concentration of monomethylatedH3-K27 may not appear to correspond directly to an increase indimethylated H3-K27. In this instance, it may be presumed, however, thata reduction in the amount or concentration of monomethylated H3-K27 is,by itself, reflective of conversion of monomethylated H3-K27 todimethylated H3-K27.

Detection of conversion of dimethylated H3-K27 to trimethylated H3-K27can be accomplished using any suitable method. In one embodiment theconversion is measured using antibodies specific for dimethylated H3-K27and trimethylated H3-K27. For example, starting amounts orconcentrations of dimethylated H3-K27 and trimethylated H3-K27 may bedetermined using appropriate antibodies specific for dimethylated H3-K27and trimethylated H3-K27. Following the combination of enzyme,substrate, and test compound, resulting amounts or concentrations ofdimethylated H3-K27 and trimethylated H3-K27 may then be determinedusing appropriate antibodies specific for dimethylated H3-K27 andtrimethylated H3-K27. The beginning and resulting amounts orconcentrations of dimethylated H3-K27 and trimethylated H3-K27 can thenbe compared. Alternatively or in addition, beginning and resultingamounts or concentrations of dimethylated H3-K27 and trimethylatedH3-K27 can then be compared to corresponding amounts of concentrationsfrom a negative control. A negative control reaction, in which no testagent is included in the assay, can be run in parallel or as ahistorical control. Results of such control reaction can optionally besubtracted from corresponding results of the experimental reaction priorto or in conjunction with making the comparison mentioned above.

A test agent is identified as an inhibitor of the Y641 mutant of EZH2when methylation of H3-K27 with the test compound is less thanmethylation of H3-K27 without the test compound. In one embodiment, atest agent is identified as an inhibitor of the Y641 mutant of EZH2 whenformation of trimethylated H3-K27 in the presence of the test compoundis less than formation of trimethylated H3-K27 in the absence of thetest compound.

An aspect of the invention is a method for identifying a selectiveinhibitor of a Y641 mutant of EZH2. In one embodiment the methodincludes combining an isolated Y641 mutant of EZH2 with a histonesubstrate, a methyl group donor (e.g., SAM), and a test compound,wherein the histone substrate comprises a form of H3-K27 selected fromthe group consisting of monomethylated H3-K27, dimethylated H3-K27, anda combination of monomethylated H3-K27 and dimethylated H3-K27, therebyforming a test mixture; combining an isolated wild-type EZH2 with ahistone substrate, a methyl group donor (e.g., SAM), and a testcompound, wherein the histone substrate comprises a form of H3-K27selected from the group consisting of monomethylated H3-K27,dimethylated H3-K27, and a combination of monomethylated H3-K27 anddimethylated H3-K27, thereby forming a control mixture; performing anassay to detect trimethylation of the histone substrate in each of thetest mixture and the control mixture; calculating the ratio of (a)trimethylation with the Y641 mutant of EZH2 and the test compound (M+)to (b) trimethylation with the Y641 mutant of EZH2 without the testcompound (M−); calculating the ratio of (c) trimethylation withwild-type EZH2 and the test compound (WT+) to (d) trimethylation withwild-type EZH2 without the test compound (WT−); comparing the ratio(a)/(b) with the ratio (c)/(d); and identifying the test compound as aselective inhibitor of the Y641 mutant of EZH2 when the ratio (a)/(b) isless than the ratio (c)/(d). In one embodiment the method furtherincludes taking into account a negative control without test compoundfor either or both of the test mixture and the control mixture.

Pharmaceutical Compositions

One or more EZH2 antagonists can be administered alone to a humanpatient or in pharmaceutical compositions where they are mixed withsuitable carriers or excipient(s) at doses to treat or ameliorate adisease or condition as described herein. Mixtures of these EZH2antagonists can also be administered to the patient as a simple mixtureor in suitable formulated pharmaceutical compositions. For example, oneaspect of the invention relates to pharmaceutical composition comprisinga therapeutically effective dose of an EZH2 antagonist, or apharmaceutically acceptable salt, hydrate, enantiomer or stereoisomerthereof; and a pharmaceutically acceptable diluent or carrier.

Techniques for formulation and administration of EZH2 antagonists may befound in references well known to one of ordinary skill in the art, suchas Remington's “The Science and Practice of Pharmacy,” 21st ed.,Lippincott Williams & Wilkins 2005.

Suitable routes of administration may, for example, include oral,rectal, or intestinal administration; parenteral delivery, includingintravenous, intramuscular, intraperitoneal, subcutaneous, orintramedullary injections, as well as intrathecal, directintraventricular, or intraocular injections; topical delivery, includingeyedrop and transdermal; and intranasal and other transmucosal delivery.

Alternatively, one may administer an EZH2 antagonist in a local ratherthan a systemic manner, for example, via injection of the EZH2antagonist directly into an edematous site, often in a depot orsustained release formulation.

In one embodiment, an EZH2 antagonist is administered by directinjection into a tumor or lymph node.

Furthermore, one may administer an EZH2 antagonist in a targeted drugdelivery system, for example, in a liposome coated with cancercell-specific antibody.

The pharmaceutical compositions of the present invention may bemanufactured, e.g., by conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in a conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active EZH2 antagonistsinto preparations which can be used pharmaceutically. Proper formulationis dependent upon the route of administration chosen.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants are used in the formulationappropriate to the barrier to be permeated. Such penetrants aregenerally known in the art.

For oral administration, the EZH2 antagonists can be formulated readilyby combining the active EZH2 antagonists with pharmaceuticallyacceptable carriers well known in the art. Such carriers enable the EZH2antagonists of the invention to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated. Pharmaceuticalpreparations for oral use can be obtained by combining the active EZH2antagonist with a solid excipient, optionally grinding a resultingmixture, and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitableexcipients include fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active EZH2 antagonist doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active EZH2 antagonists may be dissolved or suspended insuitable liquids, such as fatty oils, liquid paraffin, or liquidpolyethylene glycols. In addition, stabilizers may be added.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the EZH2 antagonists for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the EZH2antagonist and a suitable powder base such as lactose or starch.

The EZH2 antagonists can be formulated for parenteral administration byinjection, e.g., bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active EZH2 antagonists in water-soluble form.Additionally, suspensions of the active EZH2 antagonists may be preparedas appropriate oily injection suspensions. Suitable lipophilic solventsor vehicles include fatty oils such as sesame oil, or synthetic fattyacid esters, such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of the EZH2antagonists to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder form forreconstitution before use with a suitable vehicle, e.g., sterilepyrogen-free water.

The EZH2 antagonists may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases, such as cocoa butter or other glycerides.

In addition to the formulations described previously, the EZH2antagonists may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for example,subcutaneously or intramuscularly or by intramuscular injection). Thus,for example, the EZH2 antagonists may be formulated with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives (for example, as a sparingly soluble salt).

Alternatively, other delivery systems for hydrophobic pharmaceuticalEZH2 antagonists may be employed. Liposomes and emulsions are examplesof delivery vehicles or carriers for hydrophobic drugs. Certain organicsolvents such as dimethysulfoxide also may be employed. Additionally,the EZH2 antagonists may be delivered using a sustained-release system,such as semi-permeable matrices of solid hydrophobic polymers containingthe therapeutic agent. Various sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the EZH2 antagonists for a few weeks up to over 100 days.Depending on the chemical nature and the biological stability of thetherapeutic reagent, additional strategies for protein stabilization maybe employed.

The pharmaceutical compositions may also comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymers,such as polyethylene glycols.

Methods of Treatment

Provided herein are methods of treating or preventing conditions anddiseases the course of which can be influenced by modulating themethylation status of histones or other proteins, wherein saidmethylation status is mediated at least in part by the activity of EZH2.Modulation of the methylation status of histones can in turn influencethe level of expression of target genes activated by methylation, and/ortarget genes suppressed by methylation.

For example, one aspect of the invention relates to a method fortreating cancer. The method comprises the step of administering to asubject having a cancer expressing a Y641 mutant of EZH2 atherapeutically effective amount of an inhibitor of EZH2, wherein theinhibitor inhibits histone methyltransferase activity of EZH2, therebytreating the cancer. In one embodiment the inhibitor inhibits histonemethyltransferase activity of the Y641 mutant of EZH2. In one embodimentthe inhibitor selectively inhibits histone methyltransferase activity ofthe Y641 mutant of EZH2. In one embodiment the cancer is a follicularlymphoma. In one embodiment the cancer is a diffuse large B-celllymphoma (DLBCL).

An aspect of the invention relates to a method for treating cancer. Themethod comprises the steps of performing an assay to detect a Y641mutant of EZH2 in a sample comprising cancer cells from a subject havinga cancer; and administering to a subject expressing a Y641 mutant ofEZH2 a therapeutically effective amount of an inhibitor of EZH2, whereinthe inhibitor inhibits histone methyltransferase activity of EZH2,thereby treating the cancer. In one embodiment the inhibitor inhibitshistone methyltransferase activity of the Y641 mutant of EZH2. In oneembodiment the inhibitor selectively inhibits histone methyltransferaseactivity of the Y641 mutant of EZH2. In one embodiment the cancer is afollicular lymphoma. In one embodiment the cancer is a diffuse largeB-cell lymphoma (DLBCL).

Diseases such as cancers and neurological disease can be treated byadministration of modulators of protein (e.g., histone) methylation,e.g., modulators of histone methyltransferase, or histone demethylaseenzyme activity. Histone methylation has been reported to be involved inaberrant expression of certain genes in cancers, and in silencing ofneuronal genes in non-neuronal cells. Modulators described herein can beused to treat such diseases, i.e., to inhibit methylation of histones inaffected cells.

Based at least on the fact that increased histone methylation has beenfound to be associated with certain cancers, a method for treatingcancer in a subject comprises administering to the subject in needthereof a therapeutically effective amount of a compound that inhibitsmethylation or restores methylation to roughly its level in counterpartnormal cells. In one embodiment a method for treating cancer in asubject comprises administering to the subject in need thereof atherapeutically effective amount of a compound that inhibits conversionof unmethylated H3-K27 to monomethylated H3-K27 (H3-K27me1). In oneembodiment a method for treating cancer in a subject comprisesadministering to the subject in need thereof a therapeutically effectiveamount of a compound that inhibits conversion of monomethylated H3-K27(H3-K27me1) to dimethylated H3-K27 (H3-K27me2). In one embodiment amethod for treating cancer in a subject comprises administering to thesubject in need thereof a therapeutically effective amount of a compoundthat inhibits conversion of H3-K27me2 to trimethylated H3-K27(H3-K27me3). In one embodiment a method for treating cancer in a subjectcomprises administering to the subject in need thereof a therapeuticallyeffective amount of a compound that inhibits both conversion ofH3-K27me1 to H3-K27me2 and conversion of H3-K27me2 to H3-K27me3. It isimportant to note that disease-specific increase in methylation canoccur at chromatin in key genomic loci in the absence of a globalincrease in cellular levels of histone or protein methylation. Forexample, it is possible for aberrant hypermethylation at keydisease-relevant genes to occur against a backdrop of global histone orprotein hypomethylation.

Modulators of methylation can be used for modulating cell proliferation,generally. For example, in some cases excessive proliferation may bereduced with agents that decrease methylation, whereas insufficientproliferation may be stimulated with agents that increase methylation.Accordingly, diseases that may be treated include hyperproliferativediseases, such as benign cell growth and malignant cell growth (cancer).

Exemplary cancers that may be treated include lymphomas, includingfollicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL).

Other cancers include Acute Lymphoblastic Leukemia; Acute MyeloidLeukemia; Adrenocortical Carcinoma; AIDS-Related Cancers; AIDS-RelatedLymphoma; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma,Childhood Cerebral; Basal Cell Carcinoma, see Skin Cancer(non-Melanoma); Bile Duct Cancer, Extrahepatic; Bladder Cancer; BoneCancer, osteosarcoma/Malignant Fibrous Histiocytoma; Brain Stem Glioma;Brain Tumor; Brain Tumor, Cerebellar Astrocytoma; Brain Tumor, CerebralAstrocytoma/Malignant Glioma; Brain Tumor, Ependymoma; Brain Tumor,Medulloblastoma; Brain Tumor, Supratentorial Primitive NeuroectodermalTumors; Brain Tumor, Visual Pathway and Hypothalamic Glioma; BreastCancer; Bronchial Adenomas/Carcinoids; Burkitt's Lymphoma; CarcinoidTumor; Carcinoid Tumor, Gastrointestinal; Carcinoma of Unknown Primary;Central Nervous System Lymphoma, Primary; Cerebellar Astrocytoma;Cervical Cancer; Childhood Cancers; Chronic Lymphocytic Leukemia;Chronic Myelogenous Leukemia; Chronic Myelogenous Leukemia, Hairy Cell;Chronic Myeloproliferative Disorders; Colon Cancer; Colorectal Cancer;Cutaneous T-Cell Lymphoma, see Mycosis Fungoides and Sezary Syndrome;Endometrial Cancer; Esophageal Cancer; Ewing's Family of Tumors;Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; EyeCancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer;Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial; GermCell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; GestationalTrophoblastic Tumor; Glioma; Glioma, Childhood Brain Stem; Glioma,Childhood Cerebral Astrocytoma; Glioma, Childhood Visual Pathway andHypothalamic; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular(Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer,Childhood (Primary); Hodgkin's Lymphoma; Hodgkin's Lymphoma DuringPregnancy; Hypopharyngeal Cancer; Hypothalamic and Visual PathwayGlioma; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas);Kaposi's Sarcoma; Kidney (Renal Cell) Cancer; Kidney Cancer; LaryngealCancer; Leukemia; Lip and Oral Cavity Cancer; Liver Cancer, Adult(Primary); Liver Cancer, Childhood (Primary); Lung Cancer, Non-SmallCell; Lung Cancer, Small Cell; Lymphoma, Primary Central Nervous System;Macroglobulinemia, Waldenstrom's; Malignant Fibrous Histiocytoma ofBone/Osteosarcoma; Medulloblastoma; Melanoma; Merkel Cell Carcinoma;Mesothelioma; Mesothelioma, Adult Malignant; Metastatic Squamous NeckCancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome;Multiple Myeloma; Multiple Myeloma/Plasma Cell Neoplasm MycosisFungoides; Myelodysplastic Syndromes; Myelodysplastic/MyeloproliferativeDiseases; Myeloid Leukemia, Adult Acute; Myeloid Leukemia, ChildhoodAcute; Myeloproliferative Disorders, Chronic; Nasal Cavity and ParanasalSinus Cancer; Nasopharyngeal Cancer; Neuroblastoma; Non-Hodgkin'sLymphoma; Non-Hodgkin's Lymphoma During Pregnancy; Oral Cancer; OralCavity Cancer, Lip and; Oropharyngeal Cancer; Osteosarcoma/MalignantFibrous Histiocytoma of Bone; Ovarian Cancer; Ovarian Epithelial Cancer;Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; PancreaticCancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer; ParathyroidCancer; Penile Cancer; Pheochromocytoma; Pineoblastoma andSupratentorial Primitive Neuroectodermal Tumors; Pituitary Tumor; PlasmaCell Neoplasm/Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy andBreast Cancer; Prostate Cancer; Rectal Cancer; Retinoblastoma;Rhabdomyosarcoma; Salivary Gland Cancer; Sarcoma, Ewing's Family ofTumors; Sarcoma, Soft Tissue; Sarcoma, Uterine; Sezary Syndrome; SkinCancer; Skin Cancer (non-Melanoma); Small Intestine Cancer; Soft TissueSarcoma; Squamous Cell Carcinoma, see Skin Cancer (non-Melanoma);Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric)Cancer; Testicular Cancer; Thymoma; Thymoma and Thymic Carcinoma;Thyroid Cancer; Transitional Cell Cancer of the Renal Pelvis and Ureter;Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer ofUnusual Cancers of Childhood; Urethral Cancer; Uterine Cancer,Endometrial; Uterine Sarcoma; Vaginal Cancer; Visual Pathway andHypothalamic Glioma; Vulvar Cancer; Waldenstrom's Macroglobulinemia;Wilms' Tumor; and Women's Cancers.

Any other disease in which epigenetic methylation, which is mediated byEZH2, plays a role may be treatable or preventable using compounds andmethods described herein.

For example, neurologic diseases that may be treated include epilepsy,schizophrenia, bipolar disorder or other psychological and/orpsychiatric disorders, neuropathies, skeletal muscle atrophy, andneurodegenerative diseases, e.g., a neurodegenerative disease. Exemplaryneurodegenerative diseases include: Alzheimer's, Amyotrophic LateralSclerosis (ALS), and Parkinson's disease. Another class ofneurodegenerative diseases includes diseases caused at least in part byaggregation of poly-glutamine. Diseases of this class include:Huntington's Diseases, Spinalbulbar Muscular Atrophy (SBMA or Kennedy'sDisease), Dentatorubropallidoluysian Atrophy (DRPLA), SpinocerebellarAtaxia 1 (SCA1), Spinocerebellar Ataxia 2 (SCA2), Machado-Joseph Disease(MJD; SCA3), Spinocerebellar Ataxia 6 (SCA6), Spinocerebellar Ataxia 7(SCAT), and Spinocerebellar Ataxia 12 (SCA12).

Also provided herein are methods for selecting a treatment for a subjecthaving a cancer. The method includes determining responsiveness of thesubject to an EZH2 inhibitor by the dimethylated H3-K27 level,preferably by the levels of dimethylated H3-K27 and trimethlated H3-K27;and providing the EZH2 inhibitor to the subject when the subject isresponsive to the EZH2 inhibitor. In one embodiment, the cancer is afollicular lymphoma. Alternatively, the cancer is a diffuse large B-celllymphoma (DLBCL). In another preferred embodiment, the subject expressesa Y641 mutant EZH2. In a preferred embodiment, the Y641 mutant is Y641F,Y641H, Y641N or Y641S.

Combination Therapy

In one aspect of the invention, an EZH2 antagonist, or apharmaceutically acceptable salt thereof, can be used in combinationwith another therapeutic agent to treat diseases such as cancer and/orneurological disorders. For example, the additional agent can be atherapeutic agent that is art-recognized as being useful to treat thedisease or condition being treated by the compound of the presentinvention. The additional agent also can be an agent that imparts abeneficial attribute to the therapeutic composition (e.g., an agent thataffects the viscosity of the composition).

The combination therapy contemplated by the invention includes, forexample, administration of a compound of the invention, or apharmaceutically acceptable salt thereof, and additional agent(s) in asingle pharmaceutical formulation as well as administration of acompound of the invention, or a pharmaceutically acceptable saltthereof, and additional agent(s) in separate pharmaceuticalformulations. In other words, co-administration shall mean theadministration of at least two agents to a subject so as to provide thebeneficial effects of the combination of both agents. For example, theagents may be administered simultaneously or sequentially over a periodof time.

The agents set forth below are for illustrative purposes and notintended to be limiting. The combinations, which are part of thisinvention, can be the compounds of the present invention and at leastone additional agent selected from the lists below. The combination canalso include more than one additional agent, e.g., two or threeadditional agents if the combination is such that the formed compositioncan perform its intended function.

For example, one aspect of the invention relates to the use of an EZH2antagonist in combination with another agent for the treatment of cancerand/or a neurological disorder. In one embodiment, an additional agentis an anticancer agent that is a compound that affects histonemodifications, such as an HDAC inhibitor. In certain embodiments, anadditional anticancer agent is selected from the group consisting ofchemotherapetics (such as 2CdA, 5-FU, 6-Mercaptopurine, 6-TG, Abraxane™,Accutane®, Actinomycin-D, Adriamycin®, Alimta®, all-trans retinoic acid,amethopterin, Ara-C, Azacitadine, BCNU, Blenoxane®, Camptosar®, CeeNU®,Clofarabine, Clolar™, Cytoxan®, daunorubicin hydrochloride, DaunoXome®,Dacogen®, DIC, Ellence®, Eloxatin®, Emcyt®, etoposide phosphate,Fludara®, FUDR®, Gemzar®, Gleevec®, hexamethylmelamine, Hycamtin®,Hydrea®, Idamycin®, Ifex®, ixabepilone, Ixempra®, L-asparaginase,Leukeran®, liposomal Ara-C, L-PAM, Lysodren, Matulane®, mithracin,Mitomycin-C, Myleran®, Navelbine®, Neutrexin®, nilotinib, Nipent®,Nitrogen Mustard, Novantrone®, Oncaspar®, Panretin®, Paraplatin®,Platinol®, prolifeprospan 20 with carmustine implant, Sandostatin®,Targretin®, Tasigna®, Taxotere®, Temodar®, TESPA, Trisenox®, Valstar®,Velban®, Vidaza™, vincristine sulfate, VM 26, Xeloda® and Zanosar®);biologics (such as Alpha Interferon, Bacillus Calmette-Guerin, Bexxar®,Campath®, Ergamisol®, Erlotinib, Herceptin®, Interleukin-2, Iressa®,lenalidomide, Mylotarg®, Ontak®, Pegasys®, Revlimid®, Rituxan®,Tarceva™, Thalomid®, Tykerb®, Velcade® and Zevalin™); corticosteroids,(such as dexamethasone sodium phosphate, DeltaSone® and Delta-Cortef®);hormonal therapies (such as Arimidex®, Aromasin®, Casodex®, Cytadren®,Eligard®, Eulexin®, Evista®, Faslodex®, Femara®, Halotestin®, Megace®,Nilandron®, Nolvadex®, Plenaxis™ and Zoladex®); and radiopharmaceuticals(such as Iodotopet, Metastron®, Phosphocol® and Samarium SM-153).

Dosage

As used herein, a “therapeutically effective amount” or “therapeuticallyeffective dose” is an amount of an EZH2 antagonist or a combination oftwo or more such compounds, which inhibits, totally or partially, theprogression of the condition or alleviates, at least partially, one ormore symptoms of the condition. A therapeutically effective amount canalso be an amount which is prophylactically effective. The amount whichis therapeutically effective will depend upon the patient's size andgender, the condition to be treated, the severity of the condition andthe result sought. In one embodiment, a therapeutically effective doserefers to that amount of the EZH2 antagonists that results inamelioration of symptoms in a patient. For a given patient, atherapeutically effective amount may be determined by methods known tothose of skill in the art.

Toxicity and therapeutic efficacy of EZH2 antagonists can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the maximum tolerated dose (MTD) and theED₅₀ (effective dose for 50% maximal response). The dose ratio betweentoxic and therapeutic effects is the therapeutic index and it can beexpressed as the ratio between MTD and ED₅₀. The data obtained fromthese cell culture assays and animal studies can be used in formulatinga range of dosage for use in humans. Dosage may also be guided bymonitoring the EZH2 antagonist's effect on pharmacodynamic markers ofenzyme inhibition (e.g., histone methylation or target gene expression)in diseased or surrogate tissue. Cell culture or animal experiments canbe used to determine the relationship between doses required for changesin pharmacodynamic markers and doses required for therapeutic efficacycan be determined in cell culture or animal experiments or early stageclinical trials. The dosage of such EZH2 antagonists lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. In thetreatment of crises, the administration of an acute bolus or an infusionapproaching the MTD may be required to obtain a rapid response.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain themethyltransferase modulating effects, or minimal effective concentration(MEC) for the required period of time to achieve therapeutic efficacy.The MEC will vary for each EZH2 antagonist but can be estimated from invitro data and animal experiments. Dosages necessary to achieve the MECwill depend on individual characteristics and route of administration.However, high pressure liquid chromatography (HPLC) assays or bioassayscan be used to determine plasma concentrations.

Dosage intervals can also be determined using the MEC value. In certainembodiments, EZH2 antagonists should be administered using a regimenwhich maintains plasma levels above the MEC for 10-90% of the time,preferably between 30-90% and most preferably between 50-90% until thedesired amelioration of symptoms is achieved. In other embodiments,different MEC plasma levels will be maintained for differing amounts oftime. In cases of local administration or selective uptake, theeffective local concentration of the drug may not be related to plasmaconcentration.

One of skill in the art can select from a variety of administrationregimens and the amount of EZH2 antagonist administered will, of course,be dependent on the subject being treated, on the subject's weight, theseverity of the affliction, the manner of administration and thejudgment of the prescribing physician.

Compounds and Pharmaceutical Compositions

Aspects of the invention concern compounds which are useful according tothe methods of the invention. These compounds are referred to herein as“inhibitors of EZH2” and, equivalently, “EZH2 antagonists”. Thecompounds can be presented as the compounds per se, pharmaceuticallyacceptable salts of the compounds, or as pharmaceutical compositions.

Such compounds specifically include Compound 75

and pharmaceutically acceptable salts thereof.

The invention further includes a pharmaceutical composition comprisingCompound 75

or a pharmaceutically acceptable salt thereof.

An EZH2 antagonist and optionally other therapeutics can be administeredper se (neat) or in the form of a pharmaceutically acceptable salt. Whenused in medicine the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts can conveniently be used toprepare pharmaceutically acceptable salts thereof.

Compounds useful in accordance with the invention may be provided assalts with pharmaceutically compatible counterions (i.e.,pharmaceutically acceptable salts). A “pharmaceutically acceptable salt”means any non-toxic salt that, upon administration to a recipient, iscapable of providing, either directly or indirectly, a compound or aprodrug of a compound useful in accordance with this invention. A“pharmaceutically acceptable counterion” is an ionic portion of a saltthat is not toxic when released from the salt upon administration to asubject. Pharmaceutically compatible salts may be formed with manyacids, including but not limited to hydrochloric, sulfuric, acetic,lactic, tartaric, malic, and succinic acids. Salts tend to be moresoluble in water or other protic solvents than their corresponding freebase forms. The present invention includes the use of such salts.

Pharmaceutically acceptable acid addition salts include those formedwith mineral acids such as hydrochloric acid and hydrobromic acid, andalso those formed with organic acids such as maleic acid. For example,acids commonly employed to form pharmaceutically acceptable saltsinclude inorganic acids such as hydrogen bisulfide, hydrochloric,hydrobromic, hydroiodic, sulfuric and phosphoric acid, as well asorganic acids such as para-toluenesulfonic, salicylic, tartaric,bitartaric, ascorbic, maleic, besylic, fumaric, gluconic, glucuronic,formic, glutamic, methanesulfonic, ethanesulfonic, benzenesulfonic,lactic, oxalic, para-bromophenylsulfonic, carbonic, succinic, citric,benzoic and acetic acid, and related inorganic and organic acids. Suchpharmaceutically acceptable salts thus include sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate,terephathalate, sulfonate, xylenesulfonate, phenylacetate,phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate,glycolate, maleate, tartrate, methanesulfonate, propanesulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and thelike.

Suitable bases for forming pharmaceutically acceptable salts with acidicfunctional groups include, but are not limited to, hydroxides of alkalimetals such as sodium, potassium, and lithium; hydroxides of alkalineearth metal such as calcium and magnesium; hydroxides of other metals,such as aluminum and zinc; ammonia, and organic amines, such asunsubstituted or hydroxy-substituted mono-, di-, or trialkylamines;dicyclohexylamine; tributyl amine; pyridine; N-methyl,N-ethylamine;diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkylamines), such as mono-, bis-, or tris-(2-hydroxyethyl)amine,2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N-dialkyl-N-(hydroxy alkyl)-amines, such asN,N-dimethyl-N-(2-hydroxyethyl)amine, or tri-(2-hydroxyethyl)amine;N-methyl-D-glucamine; and amino acids such as arginine, lysine, and thelike.

Certain compounds useful in accordance with the invention and theirsalts may exist in more than one crystalline form (i.e., polymorph); thepresent invention includes the use of each of the crystal forms andmixtures thereof.

Certain compounds useful in accordance with the invention may containone or more chiral centers, and exist in different optically activeforms. When compounds useful in accordance with the invention containone chiral center, the compounds exist in two enantiomeric forms and thepresent invention includes the use of both enantiomers and mixtures ofenantiomers, such as racemic mixtures thereof. The enantiomers may beresolved by methods known to those skilled in the art; for example,enantiomers may be resolved by formation of diastereoisomeric saltswhich may be separated, for example, by crystallization; formation ofdiastereoisomeric derivatives or complexes which may be separated, forexample, by crystallization, gas-liquid or liquid chromatography;selective reaction of one enantiomer with an enantiomer-specificreagent, for example, via enzymatic esterification; or gas-liquid orliquid chromatography in a chiral environment, for example, on a chiralsupport (e.g., silica with a bound chiral ligand) or in the presence ofa chiral solvent. Where the desired enantiomer is converted into anotherchemical entity by one of the separation procedures described above, afurther step may be used to liberate the desired purified enantiomer.Alternatively, specific enantiomers may be synthesized by asymmetricsynthesis using optically active reagents, substrates, catalysts orsolvents, or by converting one enantiomer into the other by asymmetrictransformation.

When a compound useful in accordance with the invention contains morethan one chiral center, it may exist in diastereoisomeric forms. Thediastereoisomeric compounds may be separated by methods known to thoseskilled in the art (for example, chromatography or crystallization) andthe individual enantiomers may be separated as described above. Thepresent invention includes the use of various diastereoisomers ofcompounds useful in accordance with the invention, and mixtures thereof.Compounds useful in accordance with the invention may exist in differenttautomeric forms or as different geometric isomers, and the presentinvention includes the use of each tautomer and/or geometric isomer ofcompounds useful in accordance with the invention, and mixtures thereof.Compounds useful in accordance with the invention may exist inzwitterionic form. The present invention includes the use of eachzwitterionic form of compounds useful in accordance with the invention,and mixtures thereof.

Kits

An EZH2 antagonist may, if desired, be presented in a kit (e.g., a packor dispenser device) which may contain one or more unit dosage formscontaining the EZH2 antagonist. The pack may for example comprise metalor plastic foil, such as a blister pack. The pack or dispenser devicemay be accompanied by instructions for administration. Compositionscomprising an EZH2 antagonist of the invention formulated in acompatible pharmaceutical carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. Instructions for use may also be provided.

Also provided herein are kits comprising a plurality of methylationdetection reagents that detect the methylated H3-K27. For example, thekit includes mono-methylated H3-K27, di-methylated H3-K27 andtri-methylated H3-K27 detection reagents. The detection reagent is forexample antibodies or fragments thereof, polypeptide or aptamers. Thekit may contain in separate containers an aptamer or an antibody,control formulations (positive and/or negative), and/or a detectablelabel such as fluorescein, green fluorescent protein, rhodamine, cyaninedyes, Alexa dyes, luciferase, radiolabels, among others. Instructions(e.g., written, tape, VCR, CD-ROM, etc.) for carrying out the assay maybe included in the kit. The assay may for example be in the form of aWestern Blot analysis, Immunohistochemistry (1HC), immunofluorescence(IF) and Mass spectrometry (MS) as known in the art.

DEFINITIONS

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. All definitions, as defined andused herein, supersede dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e., “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

The terms “co-administration” and “co-administering” refer to bothconcurrent administration (administration of two or more therapeuticagents at the same time) and time varied administration (administrationof one or more therapeutic agents at a time different from that of theadministration of an additional therapeutic agent or agents), as long asthe therapeutic agents are present in the patient to some extent at thesame time.

The term “treating” as used herein refers to alleviate of at least onesymptom of the disease, disorder or condition. The term encompasses theadministration and/or application of one or more compounds describedherein, to a subject, for the purpose of providing management of, orremedy for a condition. “Treatment” for the purposes of this disclosure,may, but does not have to, provide a cure; rather, “treatment” may be inthe form of management of the condition. When the compounds describedherein are used to treat unwanted proliferating cells, includingcancers, “treatment” includes partial or total destruction of theundesirable proliferating cells with minimal destructive effects onnormal cells. A desired mechanism of treatment of unwanted rapidlyproliferating cells, including cancer cells, at the cellular level isapoptosis.

The term “preventing” as used herein includes either preventing orslowing the onset of a clinically evident disease progression altogetheror preventing or slowing the onset of a preclinically evident stage of adisease in individuals at risk. This includes prophylactic treatment ofthose at risk of developing a disease.

The term “subject” as used herein for purposes of treatment includes anyhuman subject who has been diagnosed with, has symptoms of, or is atrisk of developing a disorder. For methods of prevention the subject isany human subject. To illustrate, for purposes of prevention, a subjectmay be a human subject who is at risk of or is genetically predisposedto obtaining a disorder characterized by unwanted, rapid cellproliferation, such as cancer. The subject may be at risk due toexposure to carcinogenic agents, being genetically predisposed todisorders characterized by unwanted, rapid cell proliferation, and soon.

Except as otherwise indicated, standard methods can be used for theproduction of recombinant and synthetic polypeptides, fusion proteins,antibodies or antigen-binding fragments thereof, manipulation of nucleicacid sequences, production of transformed cells, and the like. Suchtechniques are known to those skilled in the art. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, 3rd Ed. (Cold SpringHarbor, N.Y., 2001); F. M. Ausubel et al. Current Protocols in MolecularBiology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc.,New York).

The term “EZH2 polypeptide” encompasses functional fragments of thefull-length polypeptides and functional equivalents of either of theforegoing that have substantially similar or substantially identicalamino acid sequences (at least about 75%, 80%, 85%, 90%, 95% 98% or moreamino acid sequence similarity or identity), where the functionalfragment or functional equivalent retains one or more of the functionalproperties of the native polypeptide.

By “functional” it is meant that the polypeptide (or nucleic acid) hasthe same or substantially similar activity with respect to one or moreof the biological properties of the native polypeptide (or nucleicacid), e.g., at least about 50%, 75%, 85%, 90%, 95% or 98% or more ofthe activity of the native polypeptide (or nucleic acid).

The term “modulate” (and grammatical equivalents) refers to an increaseor decrease in activity. In particular embodiments, the term “increase”or “enhance” (and grammatical equivalents) means an elevation by atleast about 25%, 50%, 75%, 2-fold, 3-fold, 5-fold, 10-fold, 15-fold,20-fold or more. In particular embodiments, the terms “decrease” or“reduce” (and grammatical equivalents) means a diminishment by at leastabout 25%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or more. In someembodiments, the indicated activity, substance or other parameter is notdetectable. Specifically provided are antagonists of EZH2.

The term “pharmacodynamic marker” refers to a molecular marker of drugresponse that can be measured in patients receiving the drug. The markershould be a direct measure of modulation of the drug target and be ableto show quantitative changes in response to dose. A potentialpharmacodynamic marker for EZH2 antagonists could be levels of histoneH3-K27 methylation in disease or surrogate tissue.

As used herein, the term “responsiveness” is interchangeable with terms“responsive”, “sensitive”, and “sensitivity”, and it is meant that asubject showing therapeutic response when administered an EZH inhibitor,e.g., tumor cells or tumor tissues of the subject undergo apoptosisand/or necrosis, and/or display reduced growing, dividing, orproliferation.

The term “control” or “reference” refers to methylation levels (e.g.,monomethylation level, dimethylation level or trimethylation level)detected in an adjacent non-tumor tissue isolated from the subject,detected in a healthy tissue from a healthy subject, or established by apathologist with standard methods in the art.

By “sample” it means any biological sample derived from the subject,includes but is not limited to, cells, tissues samples and body fluids(including, but not limited to, mucus, blood, plasma, serum, urine,saliva, and semen).

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Recombinant Five-Component PRC2 Complex

Wild-type EZH2 (GenBank Accession No. NM_(—)004456) or Tyr641 mutantswere co-expressed with wild-type AEBP2 (GenBank Accession No.NM_(—)153207), EED (GenBank Accession No. NM_(—)003797), SUZ12 (GenBankAccession No. NM_(—)015355) and RbAp48 (GenBank Accession No.NM_(—)005610) in Spodoptera frugiperda (Sf9) cells using a baculovirusexpression system. An N-terminal FLAG tag on the EED was used to purifyactive PRC2 complex from cell lysates (BPS Bioscience, catalog number51004). The purity of the final PRC2 preparations was assessed bySDS-PAGE with Coomassie blue staining.

Example 2 H3, H4 Peptide Panel

A library consisting of 44 peptides of 15 amino acids each wassynthesized by 21 Century Biochemicals (Marlboro, Mass.). This peptidepanel encompassed all of the amino acids of human histones H3 and H4with 5 residue overlaps between consecutive peptide sequences. TheN-terminus of each peptide was appended with biotin, and the C-terminiwere represented as the amide. Purity (>95%) and identity were confirmedby liquid chromatography/mass spectral analysis.

For study of the H3-K27 methylation status dependence of enzymeactivity, peptides were synthesized representing the amino acid sequenceof human H3 from residues 21-44 (H3:21-44) with lysine 27 represented asthe unmodified, mono-methylated, di-methylated or tri-methylated sidechain amine. These peptides were purchased from New England Peptide(Gardner, Mass.) with biotin appended to the C-terminus of each peptide.

Example 3 Evaluation of H3-K27 Methylation Status in Cells

The cell lines OCI-LY19 (ACC 528), KARPAS-422 (ACC 32), and WSU-DLCL2(ACC 575) were obtained from DSMZ. The cell lines DB (CRL-2289) andSU-DHL2 (CRL-2959) were obtained from ATCC. OCI-LY19, WSU-DLCL2, and DBcell lines were grown in RPMI-1640 with 10% FBS, and KARPAS-422 andSU-DHL2 cell lines were grown in RPMI-1640 plus 20% FBS. Cells weregrown to a density of 1.5−2×10⁶ cells/mL and 1×10⁷ cells were harvestedby centrifugation at 264×g, washed in ice cold PBS and lysed byresuspension in a 10× pellet volume of RIPA lysis buffer containing 50mM Tris-HCl, 15 0 mM NaCl, 0.25% DOC, 1% NP-40, and 1 mM EDTA (Millipore#20-188), plus 0.1% SDS and protease inhibitor tablets (Roche #1836153).Lysates were sonicated by 2 rounds of 10 1-second bursts at setting 3with a Misonix XL-2000 to ensure efficient histone extraction, andcleared by centrifugation at 4° C. using a bench top centrifuge at14,000 rpm for 10 minutes. Protein concentration was determined by BCAassay (Pierce). Four micrograms of each lysate was fractionated on 4-20%Tris-Glycine gel (Invitrogen), transferred to PVDF, and probed with thefollowing antibodies in Odyssey blocking buffer: mouse anti-EZH2 (CST3147; 1:2000 dilution), rabbit anti-H3-K27me3 (CST 9733; 1:10000dilution), rabbit anti-H3-K27me2 (CST 9755; 1:5000 dilution), rabbitanti-H3-K27me1 (Active Motif 39377; 1:5000 dilution), and mouseanti-Total H3 (CST 3638; 1:20000 dilution). Following primary Abincubation, membranes were probed with IRDye 800CW donkey-anti-mouse IgG(LiCOR #926-32212) or Alexa Fluor 680 goat-anti-rabbit IgG (Invitrogen#A-21076) secondary Ab and imaged using the LiCOR Odyssey system.

Example 4 Enzymology

As noted above, it had previously been concluded that thedisease-associated changes at Tyr641 resulted in loss of function withrespect to EZH2-catalyzed H3-K27 methylation. However, a presumptivereduction in the rate of H3-K27 methylation due to enzyme heterozygositywas difficult to rationalize as the basis for a malignant phenotype,especially in light of previous data indicating that overexpression ofEZH2, loss-of-function mutations in the corresponding H3-K27 demethylaseUTX, or overexpression of components of the PRC2, such as PHF19/PCL3,involved in increased H3-K27 trimethylation, all result in malignantphenotypes in specific human cancers. Morin et al. (2010) Nat Genet.42:181-5; Martinez-Garcia et al. (2010) Nat Genet. 42:100-1; Bracken etal. (2003) EMBO J22:5323-35; Kleer et al. (2003) Proc Natl Acad Sci USA100:11606-11; Varambally et al. (2002) Nature 419:624-9; Simon et al.(2008) Mutat Res 647:21-9; van Haaften et al. (2009) Nat Genet.41:521-3; Wang et al. (2004) Gene 343:69-78; Cao et al. (2008)Mol CellBiol 28:1862-72; and Sarma et al. (2008) Mol Cell Biol 28:2718-31).Therefore, the enzymology of these mutations was explored in greaterdetail.

Recombinant PRC2 complexes were prepared with WT and Tyr641 mutantversions of human EZH2 (see Example 1 above; Cao et al. (2004) Mol Cell15:57-67). Equal concentrations (nominally 8 nM, based on proteindeterminations) of each complex were initially tested for the ability tocatalyze ³H-methyl transfer from labeled S-adenosyl methionine (SAM) toan unmodified peptide representing the amino acid sequence surroundingH3-K27 (H3:21-44) or to native avian erythrocyte oligonucleosomes. Aspreviously reported (Morin et al. (2010) Nat Genet. 42:181-5), it wasfound that the WT enzyme displayed robust activity for methyl transferto this unmethylated peptidic substrate, but that none of the mutantenzymes displayed significant methyltransferase activity (FIG. 1A). Incontrast to the previously reported data and that in FIG. 1A, it wasfound that all of the mutant EZH2 constructs were activemethyltransferases against the avian nucleosome substrate (FIG. 1B). Thenucleosomes isolated from the avian natural source represent anadmixture of states of histone modification, including various states ofH3-K27 methylation as judged by Western blotting with H3-K27methylation-specific antibodies.

There are several potential explanations for the discordant activity ofthe mutant PRC2 complexes on peptide and nucleosome substrates. Onepossibility is that substrate recognition sites distal to the enzymeactive site (i.e., exosites) are important determinants of substratebinding and turnover; these sites would engage complementary recognitionelements on the nucleosome that are not available on small peptidicsubstrates. However, when E. coli-expressed, recombinant human histoneH3 was tested as a substrate for the WT and mutant PRC2 complexes, theresulting pattern of activity was identical to that seen for the peptidesubstrate; that is, the WT enzyme demonstrated robust methyltransferaseactivity against the H3 substrate, the Y641F mutant showed 7% theactivity of WT complex, and all other mutants displayed ≦1% the activityof WT complex. Hence, exosite engagement seems an unlikely explanationfor the current results. The nucleosome presents many lysine residuesbeyond H3-K27 as potential sites of methylation that would not bepresent in the small peptidic substrate. Thus, another possibility isthat mutation of Y641 alters the substrate specificity of EZH2 to resultin methylation of lysine residues other than H3-K27. This possibility isunlikely given the excellent agreement between mutant activity on smallpeptide and recombinant H3 protein substrates.

The apparent discordance between the present results and thosepreviously reported was resolved when the enzymatic activity of the WTand mutant PRC2 complexes were tested against a panel of peptidicsubstrates that represent all possible lysine (K) residues of histone H3and histone H4 (see Example 2 above). All of the enzyme forms showedsignificant activity only against peptides containing the equivalent ofresidue H3-K27. The specific activity of the mutants, however, wasgreatly reduced relative to WT in the order WT>>Y641F>Y641S˜Y641H>Y641N,again consistent with previous reported findings.

Example 5 Enzymology

To understand further the enzymatic activity of these mutants, and toreconcile the apparent discrepancy between activity against peptidic andnucleosome substrates, the ability of the enzyme forms to catalyzefurther methylation of various H3-K27 methylation states in the contextof the H3:21-44 peptide was studied. As stated above, it was found thatall of the mutant enzymes were deficient catalysts of unmodified H3-K27peptide methylation, relative to the WT enzyme. Remarkably, however, allof the mutant enzymes were found to be superior to WT enzyme incatalyzing further methylation of the mono- and especially thedi-methylated H3-K27 peptides (FIG. 2). Thus, the data suggest that theWT enzyme is most efficient in catalyzing the zero- to mono-methylationreaction. The mutant enzymes are defective in catalyzing this initialstep, but are more efficient than the WT enzyme in catalyzing thesubsequent steps leading from mono-methyl to di- and tri-methyl H3-K27.

The origins of the differential substrate specificities of WT and mutantEZH2 were explored through steady state enzyme kinetics. As summarizedin Table 1, the mutations have minimal effects on ground-state substraterecognition, as demonstrated by the similar values of K_(m) fornucleosome and of K_(1/2) for peptide substrates. In all cases thepeptidic substrates displayed sigmoidal binding behavior; hence theconcentration of peptide resulting in half-maximal velocity is reportedhere as K_(1/2) instead of the more common Michaelis constant, K_(m).Copeland (2005) Evaluation of Enzyme Inhibitors in Drug Discovery: AGuide to Medicinal Chemists and Pharmacologists, Wiley. The SAM K_(m)likewise displayed minimal variation among the enzyme forms, rangingfrom 208±50 to 304±64 nM. Instead, the differences in substrateutilization appear to have their origin in transition state recognition,as demonstrated by differences in k_(cat) values among the enzymes forvarious substrates (Table 1). As a result, the catalytic efficiency,quantified as the ratio k_(cat)/K (where K is either K_(m) or K_(1/2),depending on substrate identity; vide supra), varies between the WT andmutant enzymes for different states of H3-K27 methylation (Table 1).

TABLE 1 Steady state kinetic parameters for methylation reactionscatalyzed by PRC2 containing wild-type or Y641 mutants of EZH2.Substrate H3-K27 Methylation K k_(cat) k_(cat)/K Enzyme Status (nM) (h⁻¹× 10⁻²) (h⁻¹·nM⁻¹ × 10⁻⁴) WT 0 184 ± 10 84.0 ± 3.0 45.7 ± 3.0 1 436 ± 4265.4 ± 5.8 15.0 ± 2.0 2 178 ± 16  6.0 ± 0.3  3.4 ± 0.3 Nucleosome 141 ±31 42.6 ± 2.6 30.2 ± 6.9 Y641F 0 240 ± 19  4.8 ± 0.3  2.0 ± 0.2 1  404 ±124 15.0 ± 4.3  3.7 ± 1.6 2 191 ± 10 84.0 ± 2.8 44.0 ± 2.7 Nucleosome176 ± 19 65.4 ± 2.0 37.2 ± 4.2 Y641H 0 —^(a) — — 1 319 ± 57 28.2 ± 3.7 8.8 ± 2.0 2 148 ± 9  22.8 ± 0.9 15.4 ± 1.1 Nucleosome 140 ± 22 23.4 ±1.0 16.7 ± 2.7 Y641N 0 — — — 1 280 ± 11 23.4 ± 0.8  8.4 ± 0.4 2 157 ± 1196.0 ± 4.0 61.1 ± 5.0 Nucleosome 191 ± 34 23.4 ± 1.3 12.3 ± 2.3 Y641S 0— — — 1 249 ± 8  27.6 ± 0.8 11.1 ± 0.5 2 136 ± 8  59.4 ± 2.0 43.7 ± 3.0Nucleosome 137 ± 28 23.4 ± 1.4 17.1 ± 3.6 ^(a)Activity too low tomeasure.

Example 6 Enzymology

The steady state kinetic parameters listed in Table 1 made it possibleto calculate the expected levels of different H3-K27 methylation statesfor cells heterozygous for the various mutant EZH2 forms, relative tocells homozygous for the WT enzyme. To perform these simulations, anumber of simplifying assumptions were made: (1) that steady stateenzyme kinetics are relevant to PRC2-catalyzed H3-K27 methylation in thecellular context and that all measurements are made at the same timepoint in cell growth; (2) that the mutant and WT enzyme are expressed atequal levels in heterozygous cells and that the total EZH2 level isequal in all cells; (3) that the cellular concentration of SAM, relativeto its K_(m) is saturating and does not change among the cells; (4) thatthe cellular concentration of nucleosome, is similar to its K_(m) andlikewise does not change among cells; (5) that EZH1 catalyzedmethylation of H3-K27 was insignificant and constant among the cells;and (6) that any H3-K27 demethylase activity was also constant among thecells.

With these assumptions in place, the predictions illustrated in FIG. 3Awere obtained for relative levels of H3-K27me3 (top panel), H3-K27me2(middle panel) and H3-K27me1 (bottom panel). A clear pattern emergesfrom these simulations. The level of H3-K27me3 increases relative to WTcells for all mutant-harboring cells, ranging from a 30% increase forthe Y641H mutant to >400% for the Y641N mutant. At the same time, thelevels of H3-K27me2 decreases to <50% of WT for all of the mutants, andthe levels of H3-K27me1 are reduced by approximately half for allmutants, relative to WT.

The relative levels of the H3-K27 methylation states in B-cell lymphomacell lines that are known to be homozygous for WT EZH2 (OCI-LY19) orheterozygous for EZH2 Y641N (DB, KARPAS 422, and SU-DHL-6) or EZH2 Y641F(WSU-DLCL2) were then measured by Western blotting (FIG. 3B). Thepattern of relative H3-K27 methylation states seen in FIG. 3 b is inexcellent agreement with the results of the simulations based on invitro steady state kinetic parameters, despite the assumptions used inthe simulations and the use of a non-physiological peptide surrogate assubstrate.

Thus, increased H3-K27me3 was observed for all Y641 mutant-harboringcells relative to WT, decreased H3-K27me2 was observed for all Y641mutant-harboring cells relative to WT, and decreased H3-K27me1 wasobserved for at least two of the four mutant cell lines. Thenear-comparable levels of H3-K27me1 in WT and KARPAS 422 and SU-DHL-6cells may reflect different expression levels of WT and mutant EZH2,different contributions of EZH1, or other factors not accounted for inthe simulations. Nevertheless, the concordance between the predicted andexperimental patterns of H3-K27 methylation status is remarkable andsupports the view that enzymatic coupling between WT and mutant EZH2leads to increased H3-K27me3, thus resulting in the malignant phenotypeof cells that are heterozygous for these mutants.

Example 7 In Vitro Assays of PRC2 Methyltransferase Activity

Flashplate assay with peptide substrate. For initial comparison of WTand Y641 mutants of EZH2, biotinylated histone H3:21-44 peptidecontaining unmethylated K27 (New England Peptide), monomethylated K27(Millipore) or dimethylated K27 (Millipore) at a concentration of 800 nMwas combined with a mixture of S-adenosylmethionine-Cl (SAM) at 1,700nM, and 300 nM tritiated SAM (Perkin Elmer). This substrate combinationwas then added to the PRC2 in assay buffer (20 mM BICINE, 1 mM DTT,0.002% Tween 20, 0.005% bovine skin gelatin (BSG), pH 7.6). Reactionswere allowed to proceed for the indicated time interval and thenquenched by addition of excess cold SAM (600 μM final concentration).Quenched reaction mixtures were transferred to a streptavidin-coatedFlashplate (Perkin Elmer, catalog number SMP410), allowed to bind forone hour, and then detected on a TopCount NXT HTS scintillation andluminescence counter (Perkin Elmer). Each time point represented theaverage of six individual reactions. Steady state kinetic parameterswere determined under identical reaction conditions except that theconcentration of peptide or SAM was varied, while at saturatingconditions of the other substrate. Velocity was plotted as a function ofvaried substrate concentration and the data were fitted to theuntransformed version of the Michaelis-Menten equation or theuntransformed version of a sigmoidal kinetic equation to calculatevalues of K and k_(cat). Standard errors of fitted parameters are listedin Table 1 and were used to construct the error bars illustrated in FIG.2 panels B and C. Error associated with k_(cat)/K (Table 1) werecalculated according to standard methods of error propagation; thefractional error of k_(cat)/K was determined as:

$\begin{matrix}{{\mu \; \frac{k_{cat}}{K}} = \sqrt{\left( \frac{\mu \; k_{cat}}{k_{cat}} \right)^{2} + \left( \frac{\mu \; K}{K} \right)^{2}}} & (1)\end{matrix}$

where μk_(cat) is the standard error of k_(cat) and μK is the standarderror of K.

Filterplate assay with oligonucleosome. Chicken erythrocyteoligonucleosomes were purified as previously described. Fang et al.(2004) Methods Enzymol 377:213-26. Nucleosomes were combined with amixture of SAM and tritiated SAM, and added to PRC2 in assay buffer (20mM BICINE, 100 mM KCl, 1 mM DTT, 0.002% Tween 20, 0.005% BSG, pH 7.6).Reactions were run and quenched as above. Quenched reaction mixture wastransferred to a glass fiber filterplate (Millipore, catalog numberMSFBN6B) and washed three times with 10% trichloroacetic acid andallowed to dry. Microscint Zero (30 μL) was added and tritiumincorporation was detected on a TopCount scintillation and luminescencecounter. Steady state parameters were determined under identicalreaction conditions except that the concentration of nucleosome or SAMwas varied while at saturating conditions of the other substrate.Velocity was plotted as a function of varied substrate concentration andfitted to the untransformed version of the Michaelis-Menten equation toderive the values of K_(m) and k_(cat) as described above.

Example 8 Preparation of Compound 75 A. Preparation of Compound 37

To a solution of94(3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine(Townsend, A. P. et al. (2009) Org. Let. 11:2976-2979) (3.05 g, 9.96mmol) in DCE (250 mL) was added (9H-fluoren-9-yl)methyl(2-oxoethyl)carbamate (2.8 g, 9.96 mmol) and NaB(OAc)₃H (2.96 g, 13.95mmol), the mixture stirred for 4 h at room temperature. K₂CO₃ solutionwas added to pH at 8-9. DCM was added, the organic layer was dried withNa₂SO₄, concentrated and purified by SGC (DCM:MeOH=30:1) to give 37 (2.9g, yield: 50.9%).

B. Preparation of Compound 65

To a solution of 37 (2.9 g, 5.08 mmol) in DCE (250 mL), (S)-benzyl2-((tert-butoxycarbonyl)amino)-4-oxobutanoate (1.56 g, 5.08 mmol) andNaB(OAc)₃H (1.51 g, 7.11 mmol) were added, the mixture stirred for 4 hat room temperature. K₂CO₃ solution was added to pH at 8-9. DCM wasadded, the organic layer was dried with Na₂SO₄, concentrated andpurified with SGC (DCM: MeOH=100:1) to give 65 (2.8 g, yield: 63.9%).

C. Preparation of Compound 75

Step 1. To a solution of 65B (2.2 g, 2.55 mmol) in DCM (10 mL), Et₂NH(1.1 g, 15.3 mmol) were added, the mixture stirred for 4 h at roomtemperature. The mixture was concentrated to give crude 72 (2.2 g).

Step 2. To a stirred solution of 72 (167 mg, 0.26 mmol) in MeOH (4 mL),2-(4-chlorophenyl) acetaldehyde (40 mg, 0.26 mmol) was added and stirredat room temperature for 20 min. Then Na(OAc)₃BH (83 mg, 0.39 mmol) andHOAc (0.4 mL) was added and stirred overnight. Then NaHCO₃ (aq) wasadded and extracted with DCM (25 mL×3), washed with brine, dried withNa₂SO₄ and concentrated. The crude product was purified by preparativeTLC (DCM/MeOH=10:1) to afford 73 (30 mg, yield: 14%) as white powder.LC/MS (m/z): 779.7 [M+1]⁺.

Step 3. A mixture of 73 (30 mg, 0.038 mmol) and 10% Pd/C (15 mg) in MeOH(2 mL) was stirred at room temperature under H₂ overnight. The mixturewas filtered and the filtrate was concentrated to give crude product.The crude product was purified by preparative TLC (DCM/MeOH=8:1) toafford 74 (20 mg, yield: 69%) as white powder. LC/MS (m/z): 689.7[M+1]⁺.

Step 4. A solution of 74 (20 mg, 0.028 mmol) in 90% TFA (1 mL) wasstirred at room temperature for 1 h, and concentrated as a solid toremove TFA to give the compound 75 (TFA salt) as a colorless oil withoutpurification. LC/MS (m/z): 549.7 [M+1]⁺.

Example 9 Inhibition of EZH2 Wild-Type and Y641 Mutants by SAH

S-Adenosyl-L-homocysteine (SAH) was serially diluted 3 fold in DMSO for10 points and 1 μL was plated in a 384 well microtiter plate. Positivecontrol (100% inhibition standard) was 100 μM final concentration of SAHand negative control (0% inhibition standard) contained 1 μL of DMSO.SAH was then incubated for 30 minutes with 40 μL per well of EZH2wild-type and mutants at 8 nM in pH 7.6 assay buffer (20 mM BICINE, 100mM KCl, 1 mM DTT, 0.002% Tween 20, 0.005% BSG). A substrate mix at 10 μLper well was added which contained S-adenosylmethionine-C1 (SAM) at 150nM and tritiated SAM at 100 nM, and biotinylated oligonucleosome at 150nM in pH 7.6 assay buffer. Quenched enzyme reaction was transferred to astreptavidin-coated Flashplate (Perkin Elmer, catalog number SMP410),allowed to bind for one hour, and detected on a TopCount NXT HTS (PerkinElmer).

Results are shown in FIG. 7. IC50 values are shown in Table 2.

TABLE 2 Inhibition of WT EZH2 and Y641 mutants of EZH2 by SAH. WT Y641HY641S Y641N Y641F IC50, 0.467 0.263 0.283 0.380 4.80 μM

Example 10 Inhibition of EZH2 Wild-Type and Y641 Mutants by Compound 75

Compound 75 was serially diluted 3 fold in DMSO for 10 points and 1 μLwas plated in a 384 well microtiter plate. Positive control (100%inhibition standard) was 100 μM final concentration of SAH and negativecontrol (0% inhibition standard) contained 1 μL of DMSO. Compound 75 wasthen incubated for 30 minutes with 40 μL per well of EZH2 wild-type andmutants at 8 nM in pH 7.6 assay buffer (20 mM BICINE, 100 mM KCl, 1 mMDTT, 0.002% Tween 20, 0.005% BSG). A substrate mix at 10 μL per well wasadded which contained S-adenosylmethionine-C1 (SAM) at 150 nM andtritiated SAM at 100 nM, and biotinylated oligonucleosome at 150 nM inpH 7.6 assay buffer. Quenched enzyme reaction was transferred to astreptavidin-coated Flashplate (Perkin Elmer, catalog number SMP410),allowed to bind for one hour, and detected on a TopCount NXT HTS (PerkinElmer).

Results are shown in FIG. 8. IC50 values are shown in Table 3.

TABLE 3 Inhibition of WT EZH2 and Y641 mutants of EZH2 by Compound 75.WT Y641S Y641N Y641F Y641H IC50, 8.95 2.50 4.10 7.18 7.56 μM

Example 11 H3-K27me2/Me3 Ratios Predict Sensitivity to an EZH2 Inhibitor

Tumor cell lines heterozygous for the EZH2 (Y641) mutation displayincreased levels of H3-K27me3, the methylation state of H3-K27 thoughtto be important in tumorigenesis. Levels of the mono (H3-K27me1), di(H3-K27me2), or trimethylated (H3-K27me3) forms of H3-K27 in a panel ofcell lines that were WT for EZH2, or heterozygous for EZH2 (Y641)mutations were evaluated. Cell lines used are listed in Table 4. Themajority of lines are B-cell lymphoma lines, however two melanoma lineswere also included. IGR1 is a melanoma line that has recently been foundto contain a Y641N mutation in EZH2, and A375 cells were included as aWT EZH2 melanoma control line. FIGS. 9A and B show the results ofwestern blot analysis of histones isolated from this cell line panelprobed with antibodies recognizing H3-K27me1, H3-K27me2, or H3-K27me3.In general, global H3-K27me3 levels are higher in Y641 mutant containingcell lines than in cell lines expressing WT EZH2 exclusively. Twoexceptions are Farage and Pfeiffer cells, where H3-K27me3 levels weresimilar to those in WT lines. More striking are the dramatically lowerlevels of H3-K27me2 in EZH2 Y641 mutant cell lines relative to wild typecell lines. Little or no H3-K27me2 signal was observed in western blotof histones extracted from Y641 mutant cell lines, whereas the signalobserved with the same antibody in WT cell lines was more intense thanthat observed with the antibody specific for H3-K27me3. Overall, in WTcell lines the western blot signal with an HK27me2 antibody was higherthan the signal observed with the H3-K27me3 antibody, whereas theopposite was true in Y641 mutant cell lines. Thus the ratio ofH3-K27me3/me2 signal in Y641 lines is higher than that observed in WTlines. The one exception to this is the Pfeiffer cell line, which doesnot contain a Y641 EZH2 mutation, but has high H3-K27me3 signal, andlittle or no H3-K27me2 signal. Pfeiffer cells therefore have aH3-K27me3/me2 ratio similar to Y641 mutant cell lines.

The H3-K27 methylation state can also be examined by Mass spectrometry(MS), an independent method that does not rely on antibody reagents. TheMS analysis demonstrated that H3-K27me3 levels are higher in Y641 mutantand Pfeiffer lines than in the other WT lines, whereas the opposite istrue for H3-K27me2 levels. In the Y641 mutant and Pfeiffer lines,H3-K27me3 levels were higher than H3-K27me2 levels, whereas the oppositewas true in the other WT lines. These results are consistent with thoseobserved by western blot analysis in FIGS. 9A and B.

The differences in H3-K27 methylation state was also detected byimmunocytochemistry using antibodies to H3-K27me2 or H3-K27me3. Thisimmunohistochemistry assay is used for detecting aberrant H3-K27me2/3ratios associated with Y641 mutant EZH2 in formalin fixed paraffinembedded patient tumor tissue samples. A panel of five WT and five Y641mutant lymphoma cell line pellets were fixed and embedded in paraffinblocks and stained with anti-H3-K27me2 or H3-K27me3 antibodies. Anantibody to histone H3 was included as a positive control, since allcells should contain nuclear histone H3. FIG. 10 shows that all celllines were positive in 100% of cells for both H3-K27me3 and H3 staining.Under these conditions, no clear difference in H3-K27me3 stainingintensity was observed between WT and Y641 mutant cell lines. This mayreflect the limited dynamic range of chromogenic immunocytochemistrystaining compared to other methods of detection. However, as shown inFIG. 11, cell lines could be clearly segregated into those stainingpositive or negative for H3-K27me2. All WT cell lines, with theexception of Pfeiffer cells, stained positive for H3-K27me2, whereas allY641 mutant cell lines and Pfeiffer cells showed no staining with theH3-K27me2 antibody. These results are consistent with those obtained bywestern and MS analysis.

Without wishing to be bound by theory, the increased levels of H3-K27me3associated with the gain of function EZH2 (Y641) mutations may rendercells bearing EZH2 mutations more sensitive to small molecule EZH2inhibitors. To evaluate whether the increased H3-K27me3 and/or decreasedH3-K27me2 levels observed in Pfeiffer cells in the absence of an EZH2Y641 mutation would also correlate with sensitivity to EZH2 inhibitors,two compounds that demonstrate potent inhibition of EZH2 in biochemicalassays with IC50s of 85 and 16 nM respectively were tested. Treatment ofWSU-DLCL2 cells with either compound led to inhibition of globalH3-K27me3 levels, confirming their ability to enter cells and inhibitcellular EZH2 methyltransferase activity (FIG. 12).

The sensitivity of a panel of WT and Y641 mutant cell lines to eachcompound was evaluated in proliferation assays. Because theanti-proliferative activity of EZH2 inhibitors takes several days tomanifest, compounds were assessed in 11-day proliferation assays. FIG.13 shows representative growth curves for WT (OCI-LY19), or Y641 mutant(WSU-DLCL2) cell lines treated with the test compounds. Both compoundsdemonstrated anti-proliferative activity against WSU-DLCL2 cells, butlittle activity against OCI-LY19 cells. Inhibitor A was a more potentinhibitor of WSU-DLCL2 proliferation than Inhibitor B and this isconsistent with Inhibitor A being a more potent inhibitor of EZH2 inbiochemical assays. Proliferation assays were performed in a panel of WTand Y641 mutant lymphoma cell lines, with Inhibitor B, and day 11 IC90values were derived. FIG. 14A shows IC90 values of lymphoma cell linesgrouped by EZH2 Y641 status. Overall, Y641 mutant cell linesdemonstrated increased sensitivity to EZH2 inhibitors relative to WTcell lines, although RL and SUDHL4 cells were significantly lesssensitive than other mutant lines. Pfeiffer cells are an exception,since they are WT, but are highly sensitive to the antiproliferativeeffects of both compounds with IC90s in the low or sub-nanomolar range.Pfeiffer cells demonstrate high H3-K27me3 and low H3-K27me2 levels, andso grouping cell lines according to high H3-K27me3 and low H3-K27me2gives better discrimination of EZH2 inhibitor sensitivity as shown forInhibitor B in FIG. 14B.

Thus, high H3-K27me3 and low H3-K27me2 levels can be used to predictsensitivity to EZH2 inhibitors, independent of knowledge of mutationalstatus. The aberrant methylation ratio observed in Pfeiffer cells occursby a separate mechanism that confers dependence upon EZH2 activity.

These results demonstrates that identifying EZH2 Y641 mutations inpatient tumors and/or detecting low levels of H3-K27me2 relative toH3-K27me3 through use of techniques such as western blot, MS or IHC in apatient can be used to identify which patient will respond to EZH2inhibitor treatment.

TABLE 4 Cell lines used in this study. Cancer EZH2 Status Cell LineLymphoma: Wild Type OCI-LY19 DLBCL (Diffuse Large HT Cell B CellLymphoma) MC116 and other B-cell BC-1 Lymphoma BC-3 Pfeiffer ToledoDOHH-2 Farage SR NU-DHL-1 NU-DUL-1 Y641 Mutation SU-DHL-10 (Y641F) DB(Y641N) KARPAS 422 (Y641N) SU-DHL-6 (Y641N) WSU-DLCL-2 (Y641F) RL(Y641N) SU-DHL-4 (Y641S) Melanoma Wild Type A375 Y641 Mutation IGR-1(Y641N)

EQUIVALENTS

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

We claim:
 1. A method comprising: determining the presence of an EZH2gene mutation in a sample from a subject; and treating the subject byadministering a therapeutically effective amount of an EZH2 inhibitorbased on the presence of the EZH2 gene mutation.
 2. The method of claim1, wherein the EZH2 gene mutation is in an EZH2 nucleic acid sequenceencoding the SET domain of EZH2.
 3. The method of claim 1, wherein theEZH2 gene mutation is in an EZH2 nucleic acid sequence as defined in SEQID NO:
 7. 4. The method of claim 1, wherein the subject has a cancerselected from leukemia, melanoma, and lymphoma, or is at risk ofdeveloping a cancer selected from leukemia, melanoma, and lymphoma. 5.The method of claim 4, wherein the lymphoma is selected from the groupconsisting of Non-Hodgkin's lymphoma, follicular lymphoma and diffuselarge B-cell lymphoma (DLBCL) of germinal center B cell-like (GCB)subtype.
 6. The method of claim 1, wherein the presence of an EZH2 genemutation is determined by targeted resequencing.
 7. The method of claim6, wherein the targeted resequencing comprises amplifying at least aportion of SEQ ID NO: 7 with a PCR primer.
 8. The method of claim 1,wherein the EZH2 inhibitor inhibits the conversion of H3-K27 totrimethylated H3-K27.
 9. The method of claim 1, wherein the EZH2inhibitor selectively inhibits histone methyltransferase activity of themutant form of EZH2.
 10. The method of claim 9, where in the EZH2inhibitor is a small molecule.
 11. A method comprising: determining thepresence of an EZH2 gene mutation in a sample from a subject; andselecting, based on the presence of an EZH2 gene mutation, a therapythat includes the administration of a therapeutically effective amountof an EZH2 inhibitor.
 12. The method of claim 11, wherein the EZH2 genemutation is in an EZH2 nucleic acid sequence encoding the SET domain ofEZH2.
 13. The method of claim 12, wherein the EZH2 gene mutation is inan EZH2 nucleic acid sequence as defined in SEQ ID NO:
 7. 14. The methodof claim 11, wherein the subject has a cancer selected from leukemia,melanoma, and lymphoma, or is at risk of developing a cancer selectedfrom leukemia, melanoma, and lymphoma.
 15. The method of claim 14,wherein the lymphoma is selected from the group consisting ofNon-Hodgkin's lymphoma, follicular lymphoma and diffuse large B-celllymphoma (DLBCL) of germinal center B cell-like (GCB) subtype.
 16. Themethod of claim 15, wherein the presence of an EZH2 gene mutation isdetermined by targeted resequencing.
 17. The method of claim 16, whereinthe targeted resequencing comprises amplifying at least a portion of SEQID NO: 7 with a PCR primer.
 18. The method of claim 17, wherein the EZH2inhibitor inhibits the conversion of H3-K27 to trimethylated H3-K27. 19.The method of claim 11, wherein the EZH2 inhibitor selectively inhibitshistone methyltransferase activity of the mutant form of EZH2.
 20. Themethod of claim 19, wherein the EZH2 inhibitor is a small molecule. 21.A primer-nucleic acid complex comprising: a mutant EZH2 nucleic acidsequence, and a PCR primer that is complementary to the mutant EZH2nucleic acid sequence, wherein the mutant nucleic acid sequencecomprises an EZH2 gene mutation in a nucleic acid sequence as defined inSEQ ID NO:
 7. 22. A method comprising, amplifying a nucleic acid in asample from a subject with a primer that is complementary to a mutantEZH2 nucleic acid sequence comprising an EZH2 gene mutation in a nucleicacid sequence as defined in SEQ ID NO: 7, detecting the presence of theamplified nucleic acid, and selecting, based on the presence of theamplified nucleic acid, a therapy that includes the administration of atherapeutically effective amount of an EZH2 inhibitor, or treating thesubject by administering a therapeutically effective amount of an EZH2inhibitor based on the presence of the amplified nucleic acid.